Pleiotropic Effects of a Temperature-Sensitive Mouthless Mutation in Tetrahymena thermophila on the Excretion of Extracellular Enzymes

The mode of enzyme excretion was investigated during balanced growth in wild type Tetrahymena thermophila and in a temperature-sensitive mutant that did not form a mouth and food vacuoles at the restrictive temperature. The mutant was used to determine which of the extracellular enzymes are normally excreted by defecation. During balanced growth at the permissive temperature the excretion of enzymes was constant, and maximal in complex medium. No protease activity against azocasein was detected. Changes in the growth temperature of the wild type cells only affected the release of 3′-nucleotidase. For the mutant, however, the excretion changed markedly with increasing temperature: (a) ribonuclease, deoxyribonuclease, α-glucosidase, and β-glucosidase were detected in 0–10% of normal amounts; (b) 3′- and 5′-nucleotidase, not measured previously in Tetrahymena, were found in 10- to 14-fold of normal amounts; (c) excretion of acid phosphatase was unaffected. We therefore conclude that the extracellular enzymes (a) are released by defecation, that 3′- and 5′-nucleotidase are secreted through the membrane system, and that acid phosphatase is released by lysosomes emptied through pinocytosis. It is proposed that the lysosomes used for the formation of digestive vacuoles are different from those used for the formation of pinocytotic vacuoles and for autophagic vacuoles.     

Acid hydrolases and their Release in Food Vacuole-Less Mutants of Tetrahymena thermophila*

SYNOPSIS. Mutants (NP1 and PSJ5) of Tetrahymena thermophila strains B and D 1968 exist that are unable to construct a functional oral apparatus and form food vacuoles at 37 C but which do so normally at 30 C. Food vacuole-less cells starved in dilute salt solution released similar amounts of acid phosphatase, β-N-acetyl-glucosaminidase and ±-glucosidase activity into the medium as wildtype cells during an 8-h period. Actively growing, food vacuole-less cells had ?50% less total protein, acid phosphatase, β-N-acetyl-glucosamin-idase, and ±-glucosidase per cell than wildtype cells after 72-h growth. During this time food vacuole-less cells released significant amounts of the 3 acid hydrolases into the growth medium. For each hydrolase, the total activity released from growing, food vacuole-less cells was less, on a per cell basis, than the amount released from food vacuole formers. The proportion of the total activity secreted by the mutant and the wildtype cells was the same for acid phosphatase and β-N-acetyl-glucosaminidase and somewhat lower for ±-glucosidase. It is concluded that the release of a significant amount of acid hydrolase activity from Tetrahymena is independent of food vacuole formation and may be analogous to the secretory activity of other nonphagocytic eukaryotic cells.     

Evidence of phagotrophy in Dinophysis fortii (Dinophysiales, Dinophyceae), a dinoflagellate that causes diarrhetic shellfish poisoning (DSP)

Food vacuoles were found in one species of pho‐totrophic Dinophysis, Dinophysis fortii Pavillard, collected in Okkirai Bay. Under transmission electron microscopy, almost 70% of observed food vacuoles were characterized by membranous profiles and contained large numbers of mitochondria. The mitochondria in the food vacuole had different morphologies from those in the D. fortii cytoplasm. This indicates that these vacuoles are not autolytic accumulation bodies, but ‘true’ food vacuoles. Identification of the origin of the contents failed, but the existence of large amounts of foreign mitochondria implies that the contents in the vacuoles were derived from eukaryotic prey. Other than the observation of the food vacuoles, bacterial cells were observed in the flagellar canal. Because the flagellar canal and connecting pusule sacs had been reported to relate to macromolecule uptake, the prey organisms of D. fortii were assumed to be both eukaryotic and prokaryotic organisms.     

Cell Pairing during Mating in Tetrahymena: I. Does Phagocytosis or a Cell Surface Receptor Participate in Con A Block?1

The lectin, Concanavalin A (Con A), inhibits cell pairing during mating in Tetrahymena and binds to the surface of pairing cells via receptors concentrated around the conjugation junction. Concanavalin A is also ingested in large amounts into food vacuoles. To dispel the possibility that Con A inhibits pairing via uptake into food vacuoles or through induction of food vacuole formation and to strengthen the idea that pairing is blocked through binding of Con A to cell surface receptors, we have conducted three types of experiments: 1) attempts to inhibit pairing by feeding with nutrients and with tantalum, a non-nutritive reagent; 2) a temporal analysis of the presence of food vacuoles in mating cells fed with tantalum; and 3) analysis of the restoration of pairing following the addition of α-methyl mannoside to cells previously treated with inhibitory concentrations of Con A. The results of these studies support the idea that Con A inhibits pairing by binding to receptors located on the cell surface and not by induction of or uptake into food vacuoles. We also present evidence that cells grown in an enriched proteose peptone medium are able to pair and undergo morphogenesis more readily than cells grown in 2% proteose peptone.     

MICROTUBULE ARMS AND CYTOPLASMIC STREAMING AND MICROTUBULE BENDING AND STRETCHING OF INTERTUBULE LINKS IN THE FEEDING TENTACLE OF THE SUCTORIAN CILIATE TOKOPHRYA

Microtubules attached to the pellicle at the tips of tentacles pivot through about 140° on these attachments, splay apart, and bend along their longitudinal axes when feeding occurs. The tubules could be bending in response to pellicular contractions; active bending, sliding, or contraction of the tubules may not be involved. Intertubule links apparently prevent tubules from splaying apart at certain levels. These links are probably under tension during feeding. They stretch; they sometimes become half as thick and eight times as long as they are before feeding. Often, tubules joined together by these links also change in shape; they become slightly flattened and elliptical in cross section. Cytoplasm from the ciliate Tetrahymena is drawn down a feeding tentacle inside an invagination of the Tokophrya cell membrane from the tentacle tip. The positions of arm-bearing microtubules around such invaginations indicate that arms are involved in moving invaginations along. The edges of the perforated Tetrahymena cell membrane are "sealed" to the cell membrane of Tokophrya around each feeding tentacle tip.     

Ultrastructural Localization of Acid Phosphatase in Starved Tokophrya infusionum*

SYNOPSIS. Young organisms of Tokophrya infusionum starved for several hr, are best suited for a study of the fine structure of this organism including the distribution of its organelles. Acid phosphatase was localized by a combined electron microscopy and cytochemical approach using modified Gomori methods. The enzyme was found in small dense bodies, spheroid vesicles, missile-like bodies, rough-surfaced endoplasmic reticulum, residue and autophagic vacuoles. The small dense bodies are thought to be primary lysosomes since electron micrographs show a) a continuity between the membrane of the rough-surfaced endoplasmic reticulum and that of the dense bodies and b) a connection between the contents of both structures when the dense bodies form from the endoplasmic reticulum.     

Effect of endocytosis upon acid phosphatase activity ofTetrahymena pyriformis

Summary Increased endocytosis inTetrahymena pyriformis, produced by presenting starved cells with either peptone-yeast extract medium or killed yeast cell suspension, results in increased cellular acid phosphatase activity.Tetrahymena, grown in peptone-yeast extract medium, showed increased acid phosphatase activity after phagocytosis of yeast cells. This increase was not apparent until about one hour after presentation and was maximal at about 2.5 hours.Tetrahymena, grown on yeast suspension, showed little increase in acid phosphatase activity on presentation with peptone-yeast extract medium. These results may indicate that endocytosis, of either particles or solutes, produces an adaptive increase in acid phosphatase activity (presumably lysosomal in nature) which is related to feeding.Histochemical examination failed to localise the increase in acid phosphatase activity cellularly, but small particles, of about 1 diameter, which showed acid phosphatase activity and were presumably lysosomes were noted. Closely orientated yeast cells showed varying intensities of lead deposition, from absence to intense staining. This suggests that newly ingested yeast cells may be ingested initially in a single phagosome and that thereafter one or more lysosomes may fuse with them.     

盘基网柄菌细胞分化和凋亡中酸性磷酸酶的定位

利用电镜酶细胞化学方法,观察盘基网柄菌细胞分化和凋亡过程中酸性磷酸酶的变化。在细胞丘阶段,酶反应颗粒出现在线粒体内自噬空泡内,随着内自噬空泡的逐渐增大,线粒体内的酶反应颗粒逐渐增多,线粒体内嵴结构不断破坏,直至遍布整个空泡化的线粒体内;当细胞发育至前孢子细胞时,由于嵴结构被完全破坏,酶反应颗粒主要集中在前孢子细胞空泡的单层膜上,空泡化的线粒体内酶反应颗粒逐渐消失。在凋亡的柄细胞中,自噬泡内酶反应强烈,凋亡中期的前柄细胞的细胞核中出现酶反应颗粒,均匀分布在细胞核中,直至细胞核与自噬泡融合。在孢子细胞外被与质膜间也观察到非溶酶体酸性磷酸酶。所得结果证实:线粒体内自噬小泡具有消化功能;自噬泡内酶活性与细胞器消亡有关;细胞核中的酸性磷酸酶可能作为一种非溶酶体酸性磷酸酶参与细胞核中核蛋白的脱磷酸化过程,与发育相关基因表达有关     

Phosphatase activity in the heterotrophic dinoflagellate Pfiesteria shumwayae

The ELF-97 phosphatase substrate was used to examine phosphatase activity in four strains of the estuarine heterotrophic dinoflagellate, Pfiesteria shumwayae. Acid and alkaline phosphatase activities also were evaluated at different pH values using bulk colorimetric methods. Intracellular phosphatase activity was demonstrated in P. shumwayae cells that were actively feeding on a fish cell line and in food limited cells that had not fed on fish cells for 3 days. All strains, whether actively feeding or food limited showed similar phosphatase activities. P. shumwayae cells feeding on fish cells showed ELF-97 activity near, or surrounding, the food vacuole. Relatively small, spherical ELF-97 deposits were also observed in the cytoplasm and sometimes near the plasma membrane. ELF-97 fluorescence was highly variable among cells, likely reflecting different stages in digestion and related metabolic processes. The location of enzyme activity and supporting colorimetric measurements suggest that, as in other heterotrophic protists, acid phosphatases predominate in P. shumwayae and have a general catabolic function.     

WIDESPREAD PHAGOCYTOSIS OF CILIATES AND OTHER PROTISTS BY MARINE MIXOTROPHIC AND HETEROTROPHIC THECATE DINOFLAGELLATES1

An electron microscopic examination of large amorphous inclusions located in a variety of photosynthetic thecate dinoflagellates (Alexandrium ostenfeldii (Paulsen) Balech et Tangen, Gonyaulax diegensis Kofoid, Scrippsiella sp., Ceratium longipes (Bailey) Gran, and Prorocentrum micans Ehrenberg) and a nonphotosynthetic thecate species (Amylax sp.) revealed each inclusion to be a food vacuole, the majority of which were ingested ciliate prey. Recognizable features of these ciliates included linear arrays of basal bodies and cilia consistent with oligotrich polykinetid structure, characteristic macronuclei, chloroplasts (evidently kleptoplastids), cup-shaped starch plates, and cylindrical extrusomes. Three species contained (apparent) nonciliate prey: Scrippsiella sp., whose food vacuoles consistently contained unusual and complex extrusome-like cylindrical bodies having a distinctive six-lobed, multilayered structure; P. micans, which contained an unidentified encysted cell; and a single A. ostenfeldii cell, containing a Dinophysis sp. dinoflagellate cell. Several food vacuoles of ciliate origin had a red hue. This, together with the resemblance of A. ostenfeldii cells to planozygotes, suggests that similar structures previously identified as accumulation bodies may in fact be food vacuoles and that feeding may in some cases be associated with sexual processes.     

Trait‐mediated apparent competition in an intraguild predator–prey system

Apparent competition is of broad interest due to its effects on community structure and potential applications in agriculture, restoration, and medicine. It is well‐established that apparent competition can result from changes in predator abundance or behavior caused by interactions with alternate prey, but no previous empirical study has demonstrated that apparent competition can also result from prey‐induced changes in predator morphology. This trait‐mediated alternative mechanism of apparent competition would expand the range of conditions under which apparent competition might occur in nature and identify new ways to generate or modify apparent competition in applied contexts. Here, as a proof of concept, we show that trait‐mediated apparent competition can arise from inducible offenses and show how it operates using experiments involving three ciliates. When it feeds on Colpidium kleini, the intraguild predator Tetrahymena vorax increases in size to the extent that it can then consume Paramecium aurelia, an even larger prey. When feeding only on bacteria, however, Tetrahymena remains smaller and is unable to consume Paramecium. This trait‐mediated indirect effect leads to the predatory exclusion of Paramecium, while Tetrahymena and Colpidium coexist. Developmental expansions such as those underlying the interactions observed in our study are not limited to ciliates, such as Tetrahymena, but occur among many diverse taxa and may have a surprising degree of influence over the structure and dynamics of food webs.     

Regio- and stereo-specific nitrile hydrolysis by the nitrile hydratase from Rhodococcus AJ270

Abstract Acid phosphatase activity was measured in individual cells by determining their optical densities through a scanning confocal laser microscope. The naphthol AS-TR (3-hydroxy-2-naphtoic acid 4'-chloro-2'-methylanilide) phosphate-hexazotized para-rosanilin method was used to visualise the acid phosphatase content in the light microscope. Evidence was obtained that the amount of enzyme varied in exponential growth phase cells as the fission age increased. By comparing the acid phosphatase activity with the rate of food vacuole formation, it appeared that the amount of enzyme inside the cells decreased in early clonal life, whereas the rate of food uptake increased. It was assumed that the reduction of acid phosphatase content could lead to a more extended life of vacuoles and to a decreased membrane recycling rate. In turn, the reduced supply of membrane available for food vacuole formation could partly be responsible for the decrease of the food uptake rate observed after the initial increase.     

Electron-cytochemical demonstration of acid phosphatase and thiolacetic acid-esterase activity in granular structures of the phagotrophic slime mold,Physarum confertum Macbr.

Summary The lysosomal marker enzyme, acid phosphatase, as well as non-specific esterase could be demonstrated cytochemically in cytoplasmic granules of the slime mold, Physarum confertum Macbr. The close association of enzyme-positive granules with food vacuoles is suggestive of their participation in the hydrolytic break-down of ingested food in a manner similar to that proposed for other phagotrophic cells and organisms.     

Intracellular Bubble Formation: Differences in Gas Supersaturation Tolerances Between Tetrahymena and Euglena1

Cells of Tetrahymena pyriformis, T. thermophila, and Euglena gracilis were saturated with nitrogen gas at pressures up to 300 atm and rapidly decompressed. Damage was assessed by measuring post-decompression cell fragmentation or viability. Occurrence of intracellular bubbles was determined by cinephotomicrography performed during the decompression or by direct observations afterwards. The extreme gas supersaturations induced led to intracellular bubble formation and rupture in cells of Tetrahymena that contained food vacuoles, but only with supersaturations of 175 atm or higher; 225 atm left few cells intact. Bubbles were never observed in cells of Euglena or in Tetrahymena cells freed of food vacuoles, even when they were decompressed from substantially higher nitrogen supersaturations. Cells of Euglena were most resistant and were unaffected by supersaturations up to 250 atm.     

Feeding habits of <Emphasis Type="Italic">Bathydraco marri</Emphasis> (Pisces,Notothenioidei, Bathydraconidae) from the Ross Sea,Antarctica

In comparison with other bathydraconids, all species of the genus Bathydraco are poorly known from an ecological perspective. The diet of juvenile Bathydraco marri Norman, 1938 was studied for the first time in specimens collected in the southwestern Ross Sea during summer 1998. Fish were collected in a single otter trawl catch at 330–340 m depth. The stomach content analysis showed that this species fed exclusively on crustaceans. Overall, 20 prey taxa were identified to genus or species level. Mysids, amphipods and copepods were the most important prey in decreasing order of importance. Other prey, such as Euphausia superba, isopods and tanaids were eaten occasionally and in very small amounts. A multivariate analysis was applied to feeding data to assess ontogenetic or sex-related changes in diet. No difference was detected between sexes, whereas diet of small and large fish differed in some degree. An ontogenetic shift from small and pelagic crustaceans such as copepods to benthic–benthopelagic prey such as amphipods and mysids was observed. Relating present results with published data on physiological characteristics of B. marri, it was possible to infer their feeding behaviour and mode of life. Like other bathydraconids, this species appeared to be an inactive and sluggish fish, which relied on more or less motile benthic or epibenthic prey adopting a “sit and wait” feeding strategy. On the other hand, smaller fish seem to be more active, feeding also on pelagic prey such as copepods that can be seasonally abundant, thus reducing the intraspecific competition for food.     

Colpidium-Produced RNA as a Growth Stimulant for Tetrahymena*

SYNOPSIS. During logarithmic growth, Colpidium campylum produced a complex, heat-stable material which elevated the reproductive ate of Tetrahymena subsequently inoculated into the culture. This material stimulated only early stages in the growth of Tetrahymena—effect accompanied by an increased number and size of food vacuoles. The Colpidium product was found to be RNA, precipitable from the medium by acetone. Lipids and proteins or peptides were also present in the complex; these appeared to protect the RNA from the action of chemical and physical agents.     

Fine structural and histochemical studies on salivary glands of Peripatoides novae-zealandiae (Onychophora) with special reference to acid phosphatase distribution

The Onychophora feed on small arthropods and produce saliva when ingesting prey. Although saliva undoubtedly helps to liquefy the food its constituents have not yet been fully described. The salivary glands, two long tubes of glandular epithelium, are known to secrete a powerful protease, however, besides other enzymes and mucus. In Peripatoides novae-zealandiae there are protein-secreting cells of three types, referred to here as columnar, cuboidal and modified cells, and mucus cells. The anterior two-thirds of the gland show most cell diversity, while the posterior region consists mainly of columnar cells. These are the most numerous elements overall and they probably secrete salivary protease. In thick resin sections the granules of all protein-secreting cells stain strongly with methylene blue. Those of columnar cells are markedly uneven in size and accumulate distally, eventually filling the cytoplasm. More proximal Golgi regions may be discernible. Mucus cells are all of one type and their secretion droplets are stained lightly by methylene blue. The electron microscope shows that distal microvilli, desmosomes and septate junctions are common to all gland cells. In columnar cells, secretory material is contributed by Golgi complexes and by rough endoplasmic reticulum. Early secretory vacuoles containing dense material are seen in the concavity of Golgi regions. They are precursors to larger condensing vacuoles whose contents have a more flocculent appearance, and which may attain 3–4 μm in diameter. These evolve into secretory granules, usually of uneven texture, which are up to 2–5 μm in diameter. Histochemical tests for acid phosphatase show moderate amounts of enzyme throughout the gland. In whole mounts and sections the strongest reaction is in a band of cuboidal cells along the anterior median border. Columnar cells show a diffuse cytoplasmic reaction towards the base and sometimes distal to the nucleus, and mucus cells may also react strongly round the nucleus. Cytoplasm near the lumen shows little reaction. The secretory granules do not appear to contain active enzyme. Under the electron microscope a positive reaction for acid phosphatase is seen in lysosomal derivatives near the base and lateral periphery of gland cells. These bodies are probably autophagic vacuoles and they may contain membranous whorls and possibly old secretion granules. Acid phosphatase is involved also in the elaboration of new secretory granules in both columnar and mucus cells. Dense reaction product is found in a system of interconnected tubules and cisternae near the innermost face of the Golgi complex, which is interpreted as GERL. Acid phosphatase is present in the peripheral zone of adjacent early secretory vacuoles, and interconnections occur between GERL and secretory vacuoles. It is suggested that GERL tubules containing the enzyme may fuse with early secretory vacuoles and release acid phosphatase at their periphery. The acid phosphatase reaction is negative in large condensing vacuoles and most secretory granules. These findings are consistent with what is known from mammalian cells, including those of salivary glands.     

Localization of hormone receptors inTetrahymena

Summary It can be shown by light and electron microscopic autoradiography that¹²⁵I triiodothyronine and¹²⁵I thyroxine bind to the cell membrane ofTetrahymena. For thyroxine the finer localization is at the cilia as well as in pinocytotic and food vacuoles. Some grains also appear above the nucleus. On the other hand, insulin only binds to the membrane ofTetrahymena, and binding can be inhibited by unlabelled insulin.     

The digestion ofSaccharomyces cerevisiae byAcanthamoeba castellanii

Summary The digestion ofSaccharomyces cerevisiae byAcanthamoeba castellanii, at different times after feeding, has been examined by cytochemical techniques at electron microscope level and by measurement of yeast viability. The measurement of viability, combined with cytochemistry is presented as a novel method of examining the progress of digestion. Particular attention has been given to the temporal development of digestion.Vacuoles, probably primary lysosomes, have been identified containing acid phosphatase activity within minutes of feeding and these accumulate around and fuse with phagocytic vacuoles. Acid phosphatase levels in the digestive vacuoles appeared highest at 20 to 40 minutes. Yeast digestion was observed and yeast viability began to decline at this time. Mixing of autophagic and heterophagic material was also observed. At least half of the yeast population was still viable after 90 minutes.Our method (p-nitrophenyl phosphate) of enzyme localization has demonstrated plasma membrane associated acid phosphatase activity.     

Efficiency of Filter Feeding in Two Species of Tetrahymena*

SYNOPSIS. Rates of removal of suspended India ink particles from the surrounding medium by 2 ciliates, Tetrahymena pyriformis strain GL and Tetrahymena vorax strain V₂S, have been measured. It is evident from the results that the food vacuoles concentrate the suspended particles, clearing a volume of the surrounding suspension fluid 500 × greater than the total volume of food vacuoles made during the same period of time.