Division Cysts Research Articles

Baldinia droopii sp. nov. (Suessiales, Dinophyceae), a new species from a small rainwater rock pool near Tvärminne, south-western Finland

ABSTRACT A dinoflagellate isolated from a small rainwater rockpool in Finland was found to be a new species of Baldinia, here described as B. droopii sp. nov. This is only the third formally described Baldinia species and the second described by modern methods. The new species had a complement of generic characters that clearly affiliated it to Baldinia: presence of an internal honeycomb structure, termed a lamellar body, a ventral fibre associated with the longitudinal basal body and a pentagonal resting cyst; and the absence of an apical structure, thecal plates and trichocysts. The most distinctive species-specific characters were the presence of a resting cyst with tubiform processes, a paratabulation matching the pattern of the motile cell, and a life cycle exhibiting a diel rhythm, alternating between motile and non-motile coccoid cells. Motile cells occurred during the light period, peaking after 3–4 h of light period, while practically no motile cells occurred during the dark period. LSU rDNA sequences confirmed B. droopii as a new species, showing c. 14% sequence difference compared to B. anauniensis. Asexual reproduction occurred primarily by binary fission or via a so-called division cyst. Sexual reproduction occurred in the culture indicating that D. droopii is homothallic. The zygote may form a resistant cyst with tubiform processes, but there were indications that this stage may be by-passed. Different stages of the asexual and putative sexual reproduction were observed and documented by video recordings.

Morpho-molecular and spectroscopic characterization of the freshwater dinoflagellate Unruhdinium penardii var. robustum (Kryptoperidiniaceae, Peridiniales), blooming in the Loir River, France

A freshwater dinoflagellate formed extensive red tides in the Loir River, France in September 2018. Morphological observations using light microscopy and field emission scanning electron microscopy identified the causative organism as the“dinotom” Unruhdinium penardii var. robustum, with a tabulation of Po, x, 4ʹ, 0a, 6ʹʹ, 5c (or t+4c), 5s, 5ʹʹʹ, 2ʹʹʹʹ. The thecal plate overlap of the variety is documented for the first time. Division cysts were also observed in the plankton assemblage. Sequences obtained from the vegetative cells of small subunit (SSU) and large subunit (LSU) ribosomal DNA (rDNA) are identical to previously published sequences from Japan. This is the first unambiguous identification from French waters of this species and its variety. A SSU rDNA sequence is also reported for the diatom endosymbiont, which is close to the diatom genus Discostella. Fourier transform infrared spectroscopic analysis showed that division cysts and thecae of U. penardii var. robustum are cellulose-like, but with a higher degree of cross-linking than in microcrystalline cellulose and with a more complex macromolecular buildup.

Studies on woloszynskioid dinoflagellates VIII: life cycle, resting cyst morphology and phylogeny of Tovellia rinoi sp. nov. (Dinophyceae)

:Tovellia rinoi nov. sp. is described on the basis of light microscopic and scanning electron microscopic examination of motile cells and resting cysts and the phylogenetic analysis of partial sequences of the large subunit ribosomal rRNA gene. The species was isolated from a flooded area near Aveiro, Portugal, and followed in culture through all stages of the life cycle, including formation of vegetative cells and gametes, sexual fusion, planozygote division through division cysts and the production of resting cysts. Motile cells had an ovate-conical epicone and a truncated or slightly excavated hypocone and were a little flattened dorsoventrally. Cell length was mostly 9–19 μm, the smaller cells less pigmented and with the potential to act as gametes but also capable of vegetative multiplication. The amphiesma had three series of vesicles on the epicone, one in the cingulum and two on the hypocone and an apical line of plates (ALP) oriented almost dorsoventrally across the cell apex. One vesicle separated the ALP from the cingulum on the ventral side, two on the dorsal side. The resting cysts, showing an equatorial constriction and densely covered by hair-like spines, represented a new type within the family Tovelliaceae. The new species formed a well-supported sister group to the other Tovellia species in a LSU rDNA–derived phylogeny. Sexual fusion started with gametic contact within ‘dancing groups’ of small cells. The initial contact on the mid-ventral side of both gametes was often visible as a hyaline bridge, which apparently involved a globular structure protruding from the ventral ridge of one or both gametes. The two gametes in a fusing pair behaved differently, with one gamete rotating as it fused into its partner until gametic eyespots merged and the gametic longitudinal flagella assumed the position of the paired longitudinal flagella of the planozygote. Resting cysts developed from large cells morphologically identifiable as planozygotes, with cyst spines growing rapidly. Conversion of planozygotes into division cysts that produced motile cells was also observed. Germinating cysts produced regularly two motile cells, each apparently with a single longitudinal flagellum. Nuclear cyclosis was not detected at any stage of the life cycle.

Resting cysts of Parentocirrus hortualis Voß, 1997 (Ciliophora, Hypotrichia), with preliminary notes on encystation and various types of excystation

Resting cysts of Parentocirrus hortualis were investigated, using live observation, SEM and TEM. Processes during encystation and excystation were observed in vivo under the light microscope. During encystation, the trophic body becomes globular, the ciliature is resorbed in an anterior direction, the macronuclear nodules fuse into an elongated mass, and finally a cyst wall develops. As typical for oxytrichids, the resting cysts of P. hortualis are of the kinetosome-resorbing type and their wall is made of four layers: ectocyst, mesocyst, endocyst, and metacyst. The beginning of excystation is indicated by the formation of an excystation vacuole that helps the regenerating specimen to break the cyst wall. The excysting specimen leaves the resting cyst in a thin membrane that is gradually resorbed in the outer environment. Also two other excystation modes were observed. During the rare mode, the excystation vacuole breaks the thin membrane instead of the cyst wall that ruptures under the pressure of the body of the regenerating specimen. During the reproduction mode, the regenerating specimen divides within the resting cyst, producing two to four tomites. This is the first report of division in resting cysts of oxytrichids, but reproduction in division cysts was already described in keronopsids.

Studies on woloszynskioid dinoflagellates VII. Description of Borghiella andersenii sp. nov.: light and electron microscopy and phylogeny based on LSU rDNA

Freshwater woloszynskioid dinoflagellates were collected independently in Scotland and Portugal and found to belong to a previously unknown species of the genus Borghiella, here described as B. andersenii. The new species differs in morphology and nuclear-encoded LSU rDNA and ITS sequences from B. dodgei and B. tenuissima, the two species presently comprising the genus Borghiella. Unusual features of the new species were observed particularly during asexual reproduction, which took place in the motile stage – as in many other dinoflagellates – or in a so-called division cyst, recalling cell division in the family Tovelliaceae. Such diversity in cell division is rarely reported in dinoflagellates. Morphologically Borghiella andersenii differs from B. tenuissima in being only slightly compressed dorsoventrally whereas the latter species is flat. The slight compression is also visible in lateral view. Borghiella andersenii and B. dodgei are more challenging to discriminate but the apical structure complex is only half the length in B. andersenii compared with B. dodgei (3–4 vs 6 µm). This difference can only be accounted for in the scanning electron microscope. At the light microscopy level the epicone in B. andersenii is rounded whereas it is conical in B. dodgei. Sexual reproduction in Borghiella andersenii was homothallic by formation of planozygotes, followed by apparent resting cysts. Phylogenetic studies on woloszynskioids have recently shown that they comprise a polyphyletic assemblage, which has been divided into the three families Borghiellaceae, Tovelliaceae and Suessiaceae. New species of the three families are now being found rapidly in many parts of the world, proving that the techniques required to investigate these small, morphologically similar dinoflagellates are now in place and proving that such ‘gymnodinioids’ or ‘woloszynskioids’ comprise an often overlooked biological entity in both marine and freshwater biotopes. Based on LSU rDNA, B. andersenii is most closely related to B. tenuissima.

Porpostoma guamensis n. sp., a Philasterine Scuticociliate Associated With Brown-Band Disease of Corals

Brown band disease of coral is caused by a ciliate that consumes the tissue of the corals in the genus Acropora. We describe the ciliate associated with this disease on Guam, based on: general morphology, division stages, and ciliature observed on live and protargol-stained specimens; modification of the oral structures between divisional stages, observed on protargol-stained specimens; and some aspects of behavior in field and laboratory studies. Porpostoma guamensis n. sp. is elongate and has ciliature typical for the genus; live cells are 70-500 × 20-75 μm; the macronucleus is sausage-like, elongate but often bent, positioned centrally along the main cell axis; the oral ciliature follows a basic pattern, being composed of three adoral polykinetidal regions, as described for other species in the genus, although there is variability in the organization, especially in large cells where the three regions are not easily distinguished. Ciliates fed on coral with their oral region adjacent to the tissue, which they engulfed, leaving the coral a bare skeleton. Both zooxanthellae and nematocysts from coral occurred in the ciliates. Zooxanthellae appeared to be ingested alive but deteriorated within 2-3 days. Ciliates formed thin-walled division cysts on the coral and divided up to 3 times. Cysts formed around daughter cells within cysts. We provide some observations on the complex division pattern of the ciliate (i.e. tomont-trophont-cyst) and propose a possible complete pattern that requires further validation.

Morphology, Morphometrics, and Molecular Characterization of Bryophrya gemmea n. sp. (Ciliophora, Colpodea): Implications for the Phylogeny and Evolutionary Scenario for the Formation of Oral Ciliature in the Order Colpodida

We studied the morphology, morphometry, resting, and reproductive cysts, as well as the molecular phylogeny of Bryophrya gemmea n. sp., a colpodid ciliate that was discovered in ephemeral puddles in Idaho, northwest United States. This new species is distinguished from congeners by the irregularly pentagonal adoral organelles, four to five vestibular kineties, the single micronucleus, and one to three rows of brightly refractive protuberant interkinetal cortical granules to the right of the preoral suture. Resting cysts have two distinct membranes and an outer mucous coat. As typical for most colpodids, reproduction occurs in division cysts but details of ontogenesis are unknown. The 18S rRNA gene sequence shows only weak support for the phylogenetic relationship between Bryophrya and the bryophryid genus Notoxoma previously inferred from morphologic characters. Further, our molecular phylogenies classify bryophryids rather basal within the order Colpodida, not supporting ordinal status suggested by morphologists. Based on molecular data and morphologic characters, the colpodid genus Ilsiella is removed from the family Marynidae and placed in a new family, Ilsiellidae. Considering the molecular data, an evolutionary scenario for the formation of colpodid oral structures from a cyrtolophosidid ancestor through a bryophryid intermediate is proposed.

Observations of asexual and sexual processes in Protoperidinium depressum (Dinophyceae)

Life cycle stages of the heterotrophic dinoflagellate Protoperidinium depressum were determined for a culture isolated from the west coast of Ireland. Division, sexuality, DNA content, gamete morphology, and identity of morphologically distinct life‐history stages were revealed for the first time using a suite of morphological and molecular tools. Asexual division occurred by eleutheroschisis within a temporary, immotile cyst, yielding two daughter cells. Morphological features, including apical and antapical horns, were not fully formed upon emergence from the division cyst, but developed some time after cell division. Daughter cells were half to two‐thirds the size of parent cells and swam attached in an epitheca‐to‐hypotheca orientation before separating. Stages indicating a completed life cycle, including gametes, planozygotes, and nuclear cyclosis were observed. Small cells, believed to be gametes, formed in repeated clonal and non‐clonal re‐isolations of the culture. In addition to their small size (40–50 μm), blunt apical horn, small antapical horns, and lack of pink pigmentation, these putative gametes had a thecal plate morphology different from that described for P. depressum. The LSU rDNA sequence (D1–D3 region) of small cells was, however, the same as that of P. depressum. Gametes were never witnessed to feed, but did form fusing, isogamous pairs. When isolated to well plates, gamete pairs failed to complete fusion to form a planozygote. Thus, the morphology of the initial planozygote remains uncertain. Large cells believed to be planozygotes had “typical”P. depressum morphology, but possessed two longitudinal flagella and had 2C DNA based on DAPI. Nuclear cyclosis was documented in one such tri‐flagellated, motile cell. The cell remained motile after the completion of nuclear cyclosis, but did not progress to the next life stage. Dormant hypnozygotes were not observed.

Morphogenesis of the division and encystment process of the ciliated protozoan Colpoda minima

We have investigated the main morphological features of the asexual division and encystment process in the ciliate Colpoda minima. It is a soil colpodid ciliate with a quite variable body size. The nuclear apparatus is composed of one ellipsoidal macronucleus and one lenticular micronucleus. The somatic infraciliature is made up of 69–101 kineties, 10–16 of which are postoral. The oral infraciliature is made up of two oral polykineties, the left one consists of 45–60 short oblique rows of kinetosomes whereas the right one is formed by a row of kinetosome pairs and many short rows of kinetosomes disposed without a regular arrangement. Cell division occurs in a division cyst and produces four tomites. Stomatogenesis is merotelokinetal and involves about 16–23 stomatogenic kineties. A macronuclear extrusion process takes place during cell division. The resting cyst is included in the partial-kinetosome-resorbing (PKR) group, and its cyst wall has two layers which are derived from different cystic precursors. During encystment, macronuclear division and chromatin extrusion (in some cases) have been observed. All of these characteristics of the life cycle of C. minima are compared and discussed with respect to those of other colpodid ciliates.

LIFE CYCLE OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)1

The putatively toxic dinoflagellate Pfiesteria piscicida (Steidinger et Burkholder) has been reported to have an unusual life cycle for a free-living marine dinoflagellate. As many as 24 life cycle stages were originally described for this species. During a recent phylogenetic study in which we used clonal cultures of P. piscicida, we were unable to confirm many reported life cycle stages. To resolve this discrepancy, we undertook a rigorous examination of the life cycle of P. piscicida using nuclear staining techniques combined with traditional light microscopy, high-resolution video microscopy, EM, and in situ hybridization with a suite of fluorescently labeled peptide nucleic acid (PNA) probes. The results showed that P. piscicida had a typical haplontic dinoflagellate life cycle. Asexual division occurred within a division cyst and not by binary fission of motile cells. Sexual reproduction of this homothallic species occurred via the fusion of isogamous gametes. Examination of tanks where P. piscicida was actively feeding on fish showed that amoebae were present; however, they were contaminants introduced with the fish. Whole cell probing using in situ hybridization techniques confirmed that these amoebae were hybridization negative for a P. piscicida-specific PNA probe. Direct observations of clonal P. piscicida cultures revealed no unusual life cycle stages. Furthermore, the results of this study provided no evidence for transformations to amoebae. We therefore conclude that P. piscicida has a life cycle typical of free-living marine dinoflagellates and lacks any amoeboid or other specious stages.

The Soil Flagellate Proleptomonas faecicola: Cell Organisation and Phylogeny Suggest that the only Described Free-Living Trypanosomatid is not a Kinetoplastid but has Cercomonad Affinities

The only putative free-living trypanosomatid is Proleptomonas faecicola described first by Woodcock in 1916 as a coprophilic flagellate with striking Leptomonas-like flagellar movement but lacking a kinetoplast. P faecicola was later identified by Sandon in 1927 as a widespread non-phagotrophic inhabitant of soils. No division stages were seen by either observer. An organism conforming to Woodcock's light microscope description has been isolated from tapwater and cultivated axenically in various serum-containing media. Division has been shown to occur in an aflagellate stage enclosed in a thin cyst wall. Electron microscopy of the flagellate stage reveals that, in addition to the long locomotory flagellum, a second non-motile flagellum is present attached to the body along its entire length. The flagellate's ultrastructure lacks all the major features of the Trypanosomatidae. The several mitochondria of Proleptomonas have tubular cristae and lie between intracytoplasmic microtubules originating as a loose cone associated with the flagellar basal bodies. This cytoskeleton is much reduced in the division cyst. A comparable Proleptomonas-like flagellate with similar division cysts has been observed in soil samples from farmland. Phylogenetic analysis based on SSU rRNA gene sequences suggests that the cultured organism identified here as Proleptomonas is unrelated to the Kinetoplastida and has affinities with the Phylum Cercozoa Cavalier-Smith, even though in morphology, life cycle and mode of feeding it bears little resemblance to any member of that diverse grouping.

Vegetative and sexual reproduction in Pfiesteria spp. (Dinophyceae) cultured with algal prey, and inferences for their classification

Algal-fed clonal zoospore cultures of Pfiesteria piscicida and Pfiesteria shumwayae enabled description of certain conserved morphological and reproductive features. Common modes of reproduction (especially via division cysts) were documented in herbivorous P. piscicida and P. shumwayae using cultures fed algal prey, together with supporting photography and flow cytometric DNA measurements. Other cysts were characterized such as vacuolate cysts in starved P. piscicida cultures and temporary cysts in both species fed algal prey. This study also represents the first report of sexual reproduction in Pfiesteria spp. cultures fed algal prey rather than live fish; the first report of a technique for cell cycle synchronization for these heterotrophic dinoflagellates; and the first information on storage products of cells released from Pfiesteria reproductive cysts. Sexual reproduction in algal-fed P. piscicida clonal cultures was evidenced by fusing gametes, cells with two longitudinal flagella, and nuclear cyclosis. Both isogamous and anisogamous fusions were observed, and resulting cells with two trailing flagella (i.e., planozygotes and planomeiocytes) sometimes comprised ≥50% of the flagellated cells. These cells continued feeding activity and eventually (hours) lost their flagella and formed cysts. Nuclear cyclosis and a subsequent cell division were observed in thin-walled reproductive cysts prior to release of two flagellated cells. One gamete fusion event was also documented in 1 of 20 algal-fed clones of P. shumwayae, with an aplanozygote as the product. We obtained high cell synchrony (≥90% 1C) in the tested cultures using our preferential lysis technique and tracked the decline in lipid content of excysted zoospore populations over time. The data from this study were considered together with previous research to gain insights about relationships between Pfiesteria spp. and other heterotrophic dinoflagellates. Pfiesteria spp. should be regarded as free-living predators rather than parasites because they are prey generalists without demonstrated “host” specificity and their flagellated feeding stages are not morphologically distinct from swimming stages. Although they originally were placed within the Dinamoebales because amoebae can predominate, this study as well as other published research consistently has shown that the dominant stage varies depending on culture conditions, prey type/availability and strains. The peridinoid plate structure of each Pfiesteria species, which thus far has been conserved across culture conditions and strains, supports placement of Pfiesteria spp. within the Peridiniales. At the species level, plate structure (differing by one precingular plate) and molecular data (18S rDNA) indicate that the two Pfiesteria spp. are closely related in comparison to species grouped within other genera.

Flow cytometric determination of zoospore DNA content and population DNA distribution in cultured Pfiesteria spp. (Pyrrhophyta)

The relative cellular DNA content from 23 different clonal cultures of Pfiesteria spp. zoospores was determined using a DNA fluorochrome and flow cytometry. Significant differences between Pfiesteria piscicida and P. shumwayae were detected, both in mean zoospore DNA content and population cell cycle DNA distribution. Intraspecific differences in DNA content were found between clonal zoospore cultures established from different geographical regions. Long-term cultures (years) of P. piscicida were available for testing, and a negative correlation was observed between zoospore DNA content and time in culture. Zoospore cell cycle-related DNA distributions were also markedly different between the two species in these clonal cultures. In most cultures tested, P. piscicida zoospores exhibited bimodal DNA flow histograms with G1-S-G2+M distributions, typical of eukaryotic asynchronously cycling cells. In contrast, cultures of P. shumwayae zoospores exhibited one DNA peak distribution, indicative of synchronized cells. The data are consistent with the hypothesis that P. shumwayae zoospores are interphasic cells, and mitosis in zoospore cultures of this species predominantly occurs as benthic or adherent non-motile division cysts. Light microscopy observations of the nuclear condition of electrostatically sorted zoospores of each Pfiesteria species also support this hypothesis. If highly conserved, this disparity in modes of vegetative reproduction would ramify the population dynamics of the two Pfiesteria species.

THE LIFE HISTORY OF ALEXANDRIUM TAYLORI (DINOPHYCEAE)

The gonyaulacoid dinoflagellate Alexandrium taylori Balech is reported for the first time from Italian waters. In July 1997, nonmotile stages of this species, both temporary and sexual resting cysts, were found in surface Ionian coastal waters (Mediterranean Sea) producing localized brownish‐yellow patches. Clonal cultures were established, and the life history of A. taylori was studied in the laboratory. Asexual reproduction took place during a motile phase and produced two daughter cells remaining temporarily attached in pairs. This species exhibited isogamy. Small gametes were produced from vegetative cells through the release of a division cyst and multiple fission of the protoplast. Isogametes from the same clonal strain fused and underwent sexual reproduction, forming planozygotes that subsequently developed storage bodies and dark pigmentation. The maturation of the planozygote into hypnozygote also involved an increase in size and final shedding of flagella and theca. Hypnozygotes germinated within 15 days of their formation, and a naked planomeiocyte emerged from the archeopyle to undergo successive divisions and reestablish a haploid motile population.

Morphology, Morphogenesis and Systematic Position of the Sorocarp Forming Ciliate Sorogena Stoianovitchae Bradbury & Olive, 1980

ABSTRACT Reinvestigation of the type population of the sorocarp‐forming ciliate Sorogena stoianovitchae Bradbury & Olive, 1980 using the Fernández‐Galiano technique and various electron‐microscopy techniques (scanning electron microscopy, freeze‐fracture and ultrathin sections) expands the observations reported in the original description of the species, Sorogena stoianovitchae is a colpodid ciliate with oral ciliature consisting of 25 ciliated paroral dikinetids on the right and 3‐5 small adoral organelles on the left of an elongated and domed oral slit, resembling that of the genus Platyophrya. Sorogena stoianovitchae divides in the free swimming condition and not in a division cyst, as is the case in the colpodids sensu stricto (s. str.), e.g. Colpoda, Bresslaua. or Tillina, As shown in a detailed light‐microscopy study, morphogenesis in S. stoianovitchae is of the stomatic mode typical for certain colpodid ciliates. Based on the wealth of new information the phylogenetic position of S. stoianovitchae is discussed at some length and arguments are given in favor of the following classifications: S. stoianovitchae Bradbury & Olive, 1980 currently sole member of the family Sorogenidae Bradbury & Olive, 1980; order Sorogenida Foissner, 1985;subclassColpodiaFoissner, 1985; class Colpodea Small & Lynn, 1981. This investigation facilitates the discovery of further members of this genus reported primarily from the tropical and subtropical zone.

DIVISION SYNCHRONY IN PRIMARY CULTURES OF AN ENDOZOIC DINOFLAGELLATE1

SUMMARYDivision cysts of zooxanthellae harbored by Anthopleura elegantissima were isolated from random host‐associated populations by their preferential attachment to plastic surfaces. By selecting cysts at similar stages in the cell cycle, synchronous division, excystation, and daughter cell reencystment were obtained in a medium enriched with amino acids. The interval of DNA synthesis in a single growth cycle was determined by pulse‐labeling with radioactive thymidine. Analysis of the sequence of morphologic changes within such cycles suggested that the initial selection procedure isolates G2 cells. The transit time from first generation G2 division cysts to subsequent reentry into G2 was about 70 hr.

TILLINA MAGNA: MICRONUCLEAR NUMBER, ENCYSTMENT AND VITALITY IN DIVERSE CLONES; CAPABILITIES OF AMICRONUCLEATE RACES

The number of micronuclei was examined in 50 trophic specimens and 50 resting cysts of each of 20 clones of Tillina magna, three of which were amicronucleate. In any particular clone trophic specimens and resting cysts contained approximately equivalent mean numbers of micronuclei. In different micronucleate clones the mean number varied from 4.21 to 12.61. The mean for 1,700 specimens of 17 clones was 7.07. The number in the individuals of any particular micronucleate clone was variable; some clones showed relatively little variation, e.g., 3 to 5 micronuclei; others, considerable variation, e.g., 2 to 11 micronuclei. The smallest number observed in any micronucleate individual was 2; the largest, 16. All the clones produced normal resting cysts upon depletion of the food supply (Pseudomonas fluorescens). The cysts of different clones were equally viable and capable of excystment. Their size was unaffected by the number of micronuclei. Amicronucleate cysts showed the usual macronuclear reorganization. Hence, neither the number of micronuclei nor the absence of micronuclei affected encystment, viability and size of cysts, excystment or macronuclear reorganization. An attempt was made to maintain each clone in pure-line culture for 60 days and thereby to examine the division rate and vitality. Four clones were refractory and encysted before 60 days expired. The remaining 16 clones, including the three amicronucleate ones, survived with undiminished vigor and were discontinued. The 13 micronucleate clones produced from 149 to 176 generations during the 60-day period; the three amicronucleate clones produced 154, 164, and 172 generations, respectively. Hence, the 16 surviving clones showed slight differences in their average daily division rates, but neither the divison rate nor the vitality of these clones was correlated with variations in micronuclear number or with the absence of micronuclei. Division cysts of amicronucleate clones showed the usual macronuclear reorganization after each division of the macronucleus. The four refractory clones had high micronuclear numbers. Since conjugation is rare and endomixis and aütogamy are unknown in Tillina, it is probable that amicronucleate races arise at division by an unequal distribution of the daughter micronuclei. Some of the literature on amicronucleate ciliates and on the regeneration of various types of nucleate merozoa is reviewed. The evidence shows that the macronucleus is the indispensable nuclear element in the so-called vegetative life of the organism, whereas the micronucleus during this period appears to be a relatively passive organelle. Its chief function concerns the periodic replacement of the macronucleus and the production of new hereditary combinations. Special attention is directed to the fact that inheritance in an amicronucleate race is no less precise than in a typical micronucleate race, although the division of the macronucleus is amitotic and usually reveals no suggestion of true chromosomes. It is evident that the hereditary mechanism of amicronucleate races, and perhaps of ciliates generally, differs radically from the conventional chromosomal mechanism of metazoa.