Effects of Cations on Phagocytosis in the Ciliate Pseudomicrothorax dubius1

Various cations have been examined for their effects on phagocytosis. Media with high [Ca²⁺] and low [K⁺] favor phagocytosis, which is inhibited in media with high [K⁺], [Rb⁺], or [Ba²⁺] and low [Ca²⁺]. Microscopical observations of inhibited cells demonstrate that swimming behavior is not modified but they cannot perform the initial step of phagocytosis, attachment to food; when Ca²⁺ is added, cells attach to and ingest food, demonstrating rapid reversal of inhibition. Attachment is shown to be a linear function of the ratio [K⁺]/[Ca²⁺]^1/2 or [Rb⁺]/[Ca²⁺]^1/2 in the medium. The Ca²⁺ influx inhibitor Verapamil blocks attachment, as does La³⁺; the latter is believed to compete with Ca²⁺ for access to the Ca²⁺ channel. Likewise, treatment of cells with media containing no added Ca²⁺ inhibits attachment, and addition of 10 μM Ca²⁺ allows 90% of these cells to attach to and ingest food. The ionophore A23187, known to transport Ca²⁺ into a wide variety of cells, provokes lysosomal streaming movements typical of attachment. Based upon these observations, Ca²⁺ influx plays an essential role in attachment; K⁺ efflux also appears to be necessary since tetraethylammonium chloride blocks attachment. Treatment of cells with Tetrodotoxin, an inhibitor of Na⁺ transport, or suspending them in media containing no added Na⁺ does not affect attachment or ingestion, indicating that Na⁺ is not implicated in these processes. An hypothesis is presented which implicates Ca²⁺ in both direct adhesion and exocytosis phenomena during attachment.     

Structural and Biochemical Characterization of Isolated Trichocysts of the Ciliate Pseudomicrothorax dubius1

ABSTRACT Trichocysts of Pseudomicrothorax dubius were ejected by 15% ethanol in phosphate-buffered culture medium (CM) and purified on discontinuous sucrose gradients, in which they concentrated in the lower part of the 27% phase and in the 57% phase. These phases were washed by 15% ethanol in CM, or by CM alone, and pooled. Ejected trichocysts observed by Nomarski interference contrast microscopy and after negative-staining for electron microscopy show a shaft with periodic cross-bands and four opened-out arms, sometimes with electron-dense droplets at both ends of each arm. On SDS-PAGE, trichocysts show ?20 protein bands. The major bands are at 31 and 30 kD (G₁), 27 and 26.5 kD (G₂), 25 kD, 23 kD, and six bands at 15–20 kD (G₃). Minor bands are observed above 30 kD, among them ciliary components which contaminate the trichocyst fraction. The trichocyst banding pattern was reproducible with different ejection media; however, the 30 kD disappeared when the buffered ejection medium contained no added Ca²⁺ or contained EDTA. When the trichocyst extract is solubilized in sample buffer without 2-mercaptoethanol, the major trichocyst bands are those of G₁ and bands at 32.5–35 kD and 41 kD, which appear to be dimers of a few of the G₃ proteins. On two-dimensional gels of trichocysts, ?40 acidic protein spots are resolved with pI's of 4.6–6.6. On Western blots of two-dimensional gels, glycoproteins were revealed by Concavalin A-peroxidase labeling in three spots of G₃, in two spots at 23 kD, in all five spots of G₁, and in seven spots over 35 kD.     

The Mode of Function of the Cytopharyngeal Basket of the Ciliate Pseudomicrothorax dubius

The ciliate Pseudomicrothorax dubius feeds on filamentous blue-green algae, ingesting them at rates of up to 15 μm per second, by means of a cytopharyngeal basket. The wall of the basket is composed of 22 ± 3 nemadesmata, each of which is a bundle of about 200 microtubules which are cross-linked in a hexagonal pattern. The lumen of the non-feeding basket is filled with cytoplasma into which project the nemadesmal lamellae. Each nemadesmal lamella is attached to a nemadesm and consists of a single row of 20–30 microtubules. Each microtubule of the nemadesmal lamella bears a row of pairs of arm-like projections which are embedded in a filamentous matrix. During feeding, the lumen of the basket is occupied by the developing food vacuole. The nemadesmal lamellae are observed between the vacuole membrane and the nemadesmata, and the arms of the nemadesmal lamellae microtubules are oriented toward the membrane of the food vacuole or of small vesicles. A mechanism for the generation of force for phagocytosis by means of the microtubule arms is proposed.
During food uptake the membrane of the food vacuole increases rapidly at rates up to 270 μm² per second. Vacuole growth results from the fusion of membrane-bound vesicles. During phagocytosis a fast streaming of these vesicles can be observed in the cytoplasm surrounding the basket. The direction of streaming is opposite to that of ingestion of the algal filament. The vesicles enter the lumen of the basket at its anterior end, in a zone where the wall of the basket is perforated.     

Primary Lysosomes of the Ciliate Pseudomicrothorax dubius: Cytochemical Identification and Role in Phagocytosis*

SYNOPSIS. Filamentous cyanobacteria are ingested through the cytopharynx of the ciliate Pseudomicrothorax dubius. The cytopharynx is a complex of microtubules and microfilaments located in a highly vesiculated cytoplasm, the phagoplasm. Two types of membrane-bounded phagoplasmic vesicles can be distinguished by their differences in size, fine structure, and acid phosphatase (AcPase) content. One type has a homogeneous, electron-dense interior which is AcPase-positive. These vesicles are present in fed cells and in unfed cells devoid of food vacuoles, and thus appear to be primary lysosomes. During phagocytosis, exocytosis within the cytopharynx of the primary lysosomes results in the elaboration of a food vacuole. The vacuole grows by incorporation of lysosomal membrane; lysosomal hydrolases are liberated into the vacuole. Within less than 1 second of AcPase's entry into the food vacuole, it is detectable within the cyanobacterial cytoplasm, and within 5 seconds, destruction of the cyanobacterial filament is observed. It is hypothesized that the rapidity of hydrolase penetration of the cyanobacterial cell wall is the result of the action of molecules analogous to the “killing agents” of neutrophil leukocytes, which rapidly render bacterial envelopes permeable. AcPase, and presumably other hydrolases, are present in the cyanobacterial filament when filament destruction occurs; they thus appear implicated in this process. Hydrolases may activate an autodestruction mechanism in the cyanobacterium. Firm adherence of the food vacuole membrane to the cyanobacterial filament is demonstrated, and its role in phagocytosis is discussed.     

Electron Microscopic Localization of ATPase Activity in the Cytopharyngeal Basket of the Ciliate Pseudomicrothorax dubius

The cytopharyngeal basket of Pseudomicrothorax dubius is used to ingest filamentous blue-green algae. The basket has three main components: a sheath of microfilaments, bundles of microtubules (the nemadesmata), and ribbons of microtubules. The ribbons of microtubules (nemadesmal lamellae) are adpressed to the food vacuole during ingestion. Cytochemical techniques show that both the lamellae and the microfilamentous sheath possess ATPase activity, but the reaction product appears under different conditions in the two cases. The presence of ATPase activity within the microtubular lattices of the feeding organelle suggests the capacity for active motility. Consequently the basket seems to have two motile systems, one may be used to constrict and dilate the cytopharynx while the other is used in the inward propulsion of the forming food vacuole.     

Microtubules and Microfilaments as Major Components of a Phagocytic Apparatus: the Cytopharyngeal Basket of the Ciliate Pseudomicrothorax dubius

The cytopharyngeal basket of Pseudomicrothorax dubius , through which filamentous blue-green algae are ingested, consists of 22 (± 3) nemadesmata and nemadesmal lamellae, in the form of a tube. A cytostome, delimited by the cell membrane and surrounded by 22 (± 3) major and minor cortical corrugations, covers the end of the basket where the latter is attached to the cell cortex. Each nemadesm, at its greatest diameter, consists of about 200 microtubules which are joined together by sheet-like cross-bridges. The cross-bridges appear to be responsible for the high structural resilience of the nemadesmata. Each nemadesmal lamella is a ribbon of 20–30 microtubules, with two arm-like structures associated with one side of each microtubule. The arms are partially embedded in a fine filamentous layer. Except for a perforated zone, the wall of the basket is completely closed due to the presence of a filamentous sheath which extends between adjacent nemadesmata. Absence of the sheath allows movement of vesicles between the cytoplasm and the lumen of the basket in the perforated zone. The sheath is capable of elastic stretching during food uptake.     

Inducible defence against a ciliate grazer Pseudomicrothorax dubius,in two strains of Phormidium (cyanobacteria)

Experiments were done with two strain of filamentous, mat-forming Phormidium and their ciliate grazer Pseudomicrothorax dubius, to explain why the ciliates remain hungry in an apparent surplus of food, except for the first 24 hours after feeding. Under grazing pressure, both strains of cyanobacteria showed statistically significant increases in the number of filaments terminating in an empty sheath, compared to the control. Direct observations revealed that the mechanism behind this effect was active withdrawal of the trichomes inside the sheaths when disturbed by grazers. As P. dubius is unable to ingest trichomes with such endings, we conclude that cyanobacteria are not limited to chemical means of defence against grazers but can also defend themselves by means of movement and changes in filament morphology. This is apparently the first report on behavioural defence observed in cyanobacteria.     

Microtubules and microfilaments as major components of a phagocytic apparatus: the cytopharyngeal basket of the ciliate Pseudomicrothorax dubius.

The cytopharyngeal basket of Pseudomicrothorax dubius, through which filamentous blue-green algae are ingested, consists of 22 (+/- 3) nemadesmata and nemadesmal lamellae, in the form of a tube. A cytostome, delimited by the cell membrane and surrounded by 22 (+/- 3) major and minor cortical corrugations, covers the end of the basket where the latter is attached to the cell cortex. Each nemadesm, at its greatest diameter, consists of about 200 microtubules which are joined together by sheet-like cross-bridges. The cross-bridges appear to be responsible for the high structural resilience of the nemadesmata. Each nemadesmal lamella is a ribbon of 20--30 microtubules, with two arm-like structures associated with one side of each microtubule. The arms are partially embedded in a fine filamentous layer. Except for a perforated zone, the wall of the basket is completely closed due to the presence of a filamentous sheath which extends between adjacent nemadesmata. Absence of the sheath allows movement of vesicles between the cytoplasm and the lumen of the basket in the perforated zone. The sheath is capable of elastic stretching during food uptake.     

Demonstration of tubulin, actin and alpha-actinin by immunofluorescence in the microtubule-microfilament complex of the cytopharyngeal basket of the ciliate Pseudomicrothorax dubius

Studies were undertaken to identify cell surface markers specific for different phases of the cell cycle. Antisera were prepared in rabbits against membrane protein preparations from synchronized BW 5147 cells, an AKR mouse T-lymphoma cell line, in the G1, S, G2 or M phases of the cell cycle. These antisera were used to precipitate radioiodinated surface proteins from synchronized cells in the different phases. The immunoprecipitates were quantitatively analyzed by sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Cells in S phase had significantly higher concentrations of proteins weighing 70 × 10³ and 165 × 10³ D than cells in G1 or G2 phase. The other major labeled surface components did not vary. These results were confirmed by quantitative absorption of the antisera with synchronized cells. Comparative analysis of the antisera showed that the 165 × 10³ D peak contained at least two antigens, one recognized by both a-G1 and a-S and the other by a-G1 only. Though cells in S phase had large quantities of the 70 × 10³ D protein, intact and SDS-solubilized membrane preparations from S phase could not elicit in rabbits any antibody against that protein. These antisera did, however, have good antibody titers to the other major protein peaks and the antisera developed against cells in G1, G2 or M had good anti-70 × 10³ activity. The results suggest a qualitative molecular change in the 70 × 10³ protein during S phase.     

Reproductive Conflict and Division of Labor in Eutetramorium mocquerysi, a Myrmicine Ant Without Morphologically Distinct Female Reproductives

The myrmicine ant Eutetramorium mocquerysi Emery from Madagascar exhibits a unique social organization. All female individuals are similar in size and appearance; female reproductives with a distinct external morphology do not exist. Based on ovarian anatomy, however, two major types of females can be distinguished: females with six ovarioles and a spermatheca, which can mate and produce diploid offspring, and females with only two ovarioles, which lack a spermatheca but can lay unfertilized eggs. Individuals with three to five ovarioles are rare. Anatomical differences are not correlated with different roles. Both types of females were observed foraging, tending brood, and laying eggs. However, only females with six ovarioles and a spermatheca were the reproductively and socially most dominant individuals. Nestmate antagonism, which for the first time is demonstrated for an ant species belonging neither to the Ponerinae nor the Formicoxenini, consists of biting, antennation bouts, and ritualized dominance postures. In two colonies, removal of the dominant individual resulted in the destruction of all larvae and pupae.     

Phylogenetic Relationships of the Nassulida Within the Phylum Ciliophora Inferred from the Complete Small Subunit rRNA Gene Sequences of Furgasonia blochmanni, Obertrumia georgiana, and Pseudomicrothorax dubius

ABSTRACT. Using comparisons of complete small subunit rRNA sequences from the ciliated protozoans Furgasonia blochmanni, Obertrumia georgiana , and Pseudomicrothorax dubius we inferred the phylogenetic position of the Nassulida (Class Nassophorea) within the Ciliophora. In distance matrix analyses the Nassulida share a common ancestry with the colpodean ciliate Colpoda inflata. Distance matrix and parsimony methods convincingly demonstrate that the Nassulida plus Colpodida are members of a complex ciliate assemblage that also includes the oligohymenophorans and phyllopharyngeans. These phylogenetic inferences are largely congruent with recent analyses of 23S-like rRNA gene sequences and morphogenetic features. Groups traditionally thought to represent ancestral lineages now appear as highly derived ciliates. In contrast, heterotrichs which were considered to represent a highly evolved group, diverge at the base of the ciliates.     

An Ultrastructural and Cytochemical Study of the Cell Surface of a Suctorian Ciliate1

The surfaces of the main cell body, tentacle shaft, and knob of Discophrya collini, a freshwater suctorian ciliate, were characterized using various cytochemical techniques. Cells prepared for conventional transmission electron microscopy exhibited a 50–60 nm thick fuzzy layer over the cell body surface; this layer was absent from the tentacle knob. A thick (240 nm), two-layered surface coat surrounding the main cell body was stained with ruthenium red. This heavy coat was absent from the surface of the knob where a thin, dense, ruthenium red-positive layer and projecting filaments were present. Freeze-etched material revealed a “particle region” (150–250 nm in thickness) closely associated with the outer cell surface of the suctorian. Fixed specimens were treated with four different lectins and analyzed with electron microscopy in order to obtain information about the carbohydrate composition of the outer surface of D. collini. Concanavalin A bound to the surface of the cell body and tentacle shaft as a dense, particulate layer (80 nm thick) but thinned to 13–16 nm over the surface of the knob. Wheat germ agglutinin-treated cells also displayed a heavy, electron-dense layer (128 nm thick) that surrounded the main cell body and tentacle shaft, but only scattered patches of bound wheat germ agglutinin were observed on the surface of the knob. Discophrya treated with Helix agglutinin or peanut agglutinin appeared similar to control cells. Suctorians were treated with lectins in vivo in an attempt to inhibit capture and ingestion of their prey, Tetrahymena pyriformis, by masking prey receptor sites on the knob. Concanavalin A and, to a lesser degree, wheat germ agglutinin, successfully inhibited attachment of the prey organism. Helix agglutinin and peanut agglutinin had little effect on prey capture.     

Stimulus Control of Feeding Behavior in the Grasshopper Mouse

This study examined the stimulus control of feeding in the carnivorous grasshopper mouse, Onychomys leucogaster. Quantitative analysis of the actions involved in feeding showed that overall feeding behaviors of lab-reared and field-caught mice resembled one another but that the sequence of specific actions was variable. Responses by mice to cotton swabs dipped in solutions showed that after eating several crickets, the presence of prey odor more readily elicited a feeding response. The odor and moisture cues associated with exposed viscera were shown by the use of modified prey to strongly affect the orientation of feeding. Using modified prey without exposed viscera, tactile cues associated with the head and thorax proved to be important guides in the feeding response. Feeding orientation towards the head was independent of that towards the thorax. Together these data showed that the feeding sequence resembled a reaction chain and that much of the stereotypy of the mouse's feeding response resulted from the position and alignment of the cues that orient the feeding response.     

Encystment in the Hypotrichous Ciliate Paraurostyla weissei: Ultrastructure and Cytochemistry1

The cyst wall of Paraurostyla weissei consists of four morphologically distinct layers. It shows an ultrastructure and composition similar to that of the previously described kinetosome-resorbing cysts, and its cytoplasm displays characteristics of “urostylid-type” cysts. Therefore it is possible to consider it a “transition ciliate” between Stichotrichina and Sporadotrichina.     

NUCB2/nesfatin-1对小鼠摄食行为的调控及机制

目的:探讨NUCB2/nesfatin-1对小鼠摄食行为的调控及机制。方法:利用侧脑室埋管,免疫组化染色等方法,探讨侧脑室和外周注射nesfatin-1对小鼠摄食行为的影响。结果:侧脑室注射不同剂量nesfatin-1(0.3μg,1μg,3μg),注药后4 h夜间进食量明显减少,且呈显著剂量依赖关系(t=2.61~4.78,P0.05~0.01),侧脑室注射3μg nesfatin-1,小鼠前3小时累积摄食量明显降低(t=8.69~10.73,P0.01),且持续降低12小时(t=2.64,P0.05),同时餐间间隔时间明显延长(t=2.66,P0.05),每分钟/1-4 h进食量明显降低(t=2.63,P0.05),且进食每克食物所用时间明显增加(t=3.02,P0.05)。在下丘脑弓状核,外侧区和背内侧核均有NUCB2/nesfatin-1免疫阳性神经元表达。皮下或腹腔注射nesfatin-1,小鼠进食量和进食行为均无显著改变(P0.05)。结论:中枢nesfatin-1可抑制小鼠摄食行为。     

Mitosis of Micronuclei During Division and Regeneration in the Ciliate Stentor coeruleus1

Stages of mitosis of the micronuclei of Stentor coeruleus were described as seen by transmission electron microscopy. Cells in division and those regenerating new oral membranelles were studied. Microtubules were found in early prophase in the karyoplasm and interspersed between the condensing chromatin. A monaxial intranuclear spindle is formed by early metaphase, with kinetochore microtubule attachment sites on the chromosomes. The spindle elongates, separating the daughter nuclei at anaphase. A new nuclear envelope, consisting of two unit membranes, begins to form at late anaphase. Small segments of membrane found in the space between the newly forming and the old micronuclear envelopes appear to fuse to form the new nuclear envelope. No ultrastructural differences were found in the mitotic nuclei of cells in division or regeneration.     

Genetic Characterization and Use of a Restriction Fragment Length Variant in the Hypotrichous Ciliate Euplotes crassus1

Two forms of a macronuclear DNA molecule differing in the presence or absence of a restriction endonuclease recognition site have been detected in the hypotrichous ciliate Euplotes crassus. Through a series of genetic crosses the two forms were shown to be allelic, being derived from a single micronuclear genetic locus. This restriction fragment length variant (RFLV) was used as a genetic marker to determine that the migratory and stationary pronuclei generated during mating can be genetically non-identical. In addition, the RFLV was used to investigate the efficiency of processing of the alternate alleles during macronuclear development and their subsequent transmission during vegetative growth. Little or no bias in the processing and/or amplification of the two alleles was observed during macronuclear development. During vegetative growth, however, changes in the relative amounts of the two alleles were observed.