DNA fluorescent stain accumulates in the Golgi but not in the kinetosomes of amitochondriate protists.

Hindgut symbiotic trichomonads (uninucleate Caduceia versatilis, and multinucleate Stephanonympha sp. and Snyderella tabogae) from the dry-wood-eating termite Cryptotermes cavifrons (Kalotermitidae) accumulate DAPI (4,6diamidino-2-phenylindole) in the membranous sacs of the Golgi complex. This form of Golgi complex, typical of protists in the class Parabasalia, is called a parabasal body. Trichomonads contain organellar systems, mastigonts, that consist of four undulipodia (e.g. eukaryotic flagella and cilia), axostylar microtubules, a parabasal body and other structures. These cells bear from one (in the case of Caduceia) to hundreds (in the case of Snyderella) of mastigonts. These features are characteristic of their protist class (Parabasalia). The nuclei of all three species stained with DNA-specific stains: DAPI, SYTOX, acridine orange, propidium iodide, ethidium bromide and Feulgen, at optimal concentrations, but kinetosomes failed to stain at all. The nuclei, parabasal bodies and symbiotic bacteria (but no microtubular structures) fluoresced in glutaraldehyde-fixed cells stained with 1.45 microM DAPI. Parabasal bodies of Snyderella and Caduceia treated to remove lipids with Triton X-100, or treated with 5% trichloroacetic acid, lacked DAPI-fluorescence. I conclude that DNA, present as expected in nuclei and bacterial symbionts, is absent from and not associated with calonymphid kinetosomes. The reason for DNA-RNA stain accumulation in the Golgi cistemae is not clear.     

The motility symbiont of the termite gut flagellate Caduceia versatilis is a member of the "Synergistes" group

The flagellate Caduceia versatilis in the gut of the termite Cryptotermes cavifrons reportedly propels itself not by its own flagella but solely by the flagella of ectosymbiotic bacteria. Previous microscopic observations have revealed that the motility symbionts are flagellated rods partially embedded in the host cell surface and that, together with a fusiform type of ectosymbiotic bacteria without flagella, they cover almost the entire surface. To identify these ectosymbionts, we conducted 16S rRNA clone analyses of bacteria physically associated with the Caduceia cells. Two phylotypes were found to predominate in the clone library and were phylogenetically affiliated with the "Synergistes" phylum and the order Bacteroidales in the Bacteroidetes phylum. Probes specifically targeting 16S rRNAs of the respective phylotypes were designed, and fluorescence in situ hybridization (FISH) was performed. As a result, the "Synergistes" phylotype was identified as the motility symbiont; the Bacteroidales phylotype was the fusiform ectobiont. The "Synergistes" phylotype was a member of a cluster comprising exclusively uncultured clones from the guts of various termite species. Interestingly, four other phylotypes in this cluster, including the one sharing 95% sequence identity with the motility symbiont, were identified as nonectosymbiotic, or free-living, gut bacteria by FISH. We thus suggest that the motility ectosymbiont has evolved from a free-living gut bacterium within this termite-specific cluster. Based on these molecular and previous morphological data, we here propose a novel genus and species, "Candidatus Tammella caduceiae," for this unique motility ectosymbiont of Caducaia versatilis.     

Ultrastructure of the Parabasalid Protist Holomastigotoides

ABSTRACT. The ultrastructure of two species of Holomastigotoides is presented. The basic unit of organization of these large cells is the flagellar band. Each flagellar band consists of a row of flagellar basal bodies linked by three fiber systems. The number of flagellar bands is species dependent. The flagellar bands originate at the cell apex and are arranged in parallel spirals of increasing gyre, thus defining the conical shape of the cell. In the cell apex a striated root called a parabasal fiber is juxtaposed with the basal bodies of each flagellar band. Linear extensions of two parabasal fibers function as the spindle poles for the persistent extra-nuclear spindle. The nucleus is in close contact with the spindle poles and spindle microtubules. Parallel sheets of microtubules which constitute axostyles are nucleated along the underside of the parabasal fibers. The axostyles extend away from the cell apex, with many reaching the basal region of the cell. Some of the axostyles follow the spiral pattern of the flagellar bands. Numerous Golgi bodies are spaced regularly along the flagellar bands. Together the parabasal fiber, axostyles and Golgi bodies associated with a flagellar band are termed a parabasal complex.     

Regulation of cell volume and ion concentrations in a Halobacterium.

Changes in cell volume and ion content of a Halobacterium species are described in terms of the NaCl concentration (0.5--3.5M) and pH(4-8) of the suspending medium. Cell volume, per unit content of protein of bacteria in stationary phase cultures, rose as the [NaCl] of the growth medium was increased. Logarithmic-phase bacteria shrank as the pH fell from 7 to 5.5. These changes are characteristic of bacteria with a moderate or rapid rate of O2 consumption. Starving (i.e. nonmetabolizing) bacteria, on the other hand, did not change in size within the above ranges of [NaCl] and pH. At lower values, however, such bacteria swelled and eventually lysed. Effects of low pH on cell ions are compared in metabolizing and starving bacteria, and it is shown that changes in the state of the cell K are correlated with movements of cell Na. It appears that the cell K is used to maintain cell [Na] below the NaCl concentration of the medium. The results are explained in terms of a model involving interactions between polyelectrolytes, salts and water in the concentrated cytoplasm of these halophilic organisms.     

Ca/Ba/Sr-induced conformational changes of ciliary axonemes

Macrocilia of the ctenophore Bero? undergo Ca/Ba/Sr-dependent activation of ciliary beating and microtubule sliding disintegration [Tamm, J. Comp. Physiol. A163:23-31, 1988a; Tamm, Cell Motil. Cytoskeleton 11:126-138, 1988b; Tamm, Cell Motil. Cytoskeleton 12:104-112, 1989; Tamm and Tamm, Proc. Natl. Acad. Sci. U.S.A. 86:6987-6991, 1989]. Here we report that detergent-extracted macrocilia show an ATP-independent conformational change in response to high concentrations of Ca, Ba, or Sr ions. When applied locally by iontophoresis, these ions induce a rapid planar curvature of the distal end of the macrociliary shaft, followed by a slower relaxation to the rest position. Tip curling occurs in a direction opposite to the physiological Ca/Ba/Sr response. When applied uniformly in the bath, a threshold concentration of 10(-1) M Sr is required to induce curling of the tip, and the distal ends remain curved. Calmodulin antagonists do not inhibit the tip curling response. Previous workers found that Ca induces changes in the helical shape of isolated doublet microtubules [Miki-Noumura and Kamiya, Exp. Cell Res. 97:451-453, 1976; Miki-Noumura and Kamiya, J. Cell Biol. 81:355-360, 1979; Takasaki and Miki-Noumura, J. Mol. Biol. 158:317-324, 1982] and sperm axonemes [Okuno and Brokaw, Cell Motil. 1:349-362, 1981] and suggested that conformational changes in microtubules may play a role in Ca regulation of ciliary motility. We propose that the Ca/Ba/Sr-induced curling of the macrociliary tip is due to similar helical changes of doublet microtubules, some of which in macrocilia are prevented from sliding by permanent connections (compartmenting lamellae) between adjacent axonemes within the shaft. Although the tip curling response does not appear to be directly relevant to the physiological Ca response of macrocilia, it provides a novel system for investigating Ca-induced conformational changes of microtubules independent of dynein-powered active sliding.     

Lipid biosynthesis in synchronized cultures of the photosynthetic bacterium Rhodopseudomonas sphaeroides.

Lipid biosynthesis has been studied in photosynthetic cultures of Rhodopseudomonas sphaeroides that had been synchronized by stationary-phase cycling or by a centrifugation selection procedure. Synchrony index values in the range 0.70-0.80 were obtained for the first cell cycle with both synchronization methods. The major membrane lipids phosphatidylethanolamine and phosphatidylglycerol were accumulated discontinuously during the cell cycle, their mass doubling immediately before cell division. This accumulation of lipid corresponded to peaks in incorporation of radioactivity from either [1-14C]acetate or [2-3H]glycerol into individual acyl lipids as measured in individual portions of bacteria. For phosphatidylglycerol an additional peak of incorporation of radioactivity from [2-3H]glycerol was found midway through the cell cycle. In spite of their rather similar endogenous fatty acid compositions, the individual phosphoacylglycerols showed distinctive patterns of incorporation of radioactivity from [1-14C]acetate into their acyl moieties. The discontinuous synthesis of acyl lipids observed in cultures of Rhodopseudomonas sphaeroides synchronized by either stationary-phase cycling or centrifugation selection procedures contrasted with the accumulation of chlorophyll-protein complexes whose amounts were found to increase throughout the cell cycle. The implications of these findings for the control of lipid synthesis in bacterial photosynthetic membranes are discussed.     

Devescovinid features, a remarkable surface cytoskeleton, and epibiotic bacteria revisited in Mixotricha paradoxa, a parabasalid flagellate

Summary. This work reports on the flagellate systematics and phylogeny, cytoskeleton, prokaryote–eukaryote cell junction organisation, and epibiotic bacteria identification. It confirms the pioneer 1964 study on Mixotricha paradoxa and supplies new information. Mixotricha paradoxa has a cresta structure specific to devescovinid parabasalid flagellates, a slightly modified recurrent flagellum, and an axostylar tube containing two lamina-shaped parabasal fibres. However, many parabasal profiles are distributed throughout the cell body. There is a conspicuous cortical microfibrillar network whose strands are related to cell junction structures subjacent to epibiotic bacteria. The supposed actin composition of this network could not be demonstrated with anti-actin antibodies or phalloidin labelling. Four types of epibiotic bacteria were described. Bacillus-shaped bacteria with a Gram-negative organisation are nested in alternate rows on most of the surface of the protozoon. They induce a striated calyxlike junction structure beneath the adhesion zone linked to the cortical microfibrillar network. Slender spirochetes are attached by one differentiated end to the plasma membrane of the protozoon, forming knobs on the cell surface. Two very similar long rod-shaped bacteria are also attached on the knobs of the plasma membrane. A large spirochete attributed to the genus Canaleparolina is also attached to the protozoon. Observations on epibiotic bacteria and of their attachments are compared with several described epibiotic bacteria of symbiotic protozoa and with the results of the molecular identification of the epibiotic bacteria of M. paradoxa.Correspondence and reprints: Biologie des Protistes, Université Blaise Pascal de Clermont-Ferrand, Campus des Cézeaux, 63177 Aubiere Cedex, France.     

Total water content in different areas of the human term placenta.

The authors study the total water content in two different areas (parabasal and subchorial), in 40 normal, full-term placentas. The water content is found to be different in these two areas. As an average, it is 0.90% higher in the parabasal area. This difference persists even after washing off the fetal placental blood by perfusión with saline. The difference in the total water content between both areas, which is added to other differences previously demonstrated, must be related to a different functional role of the placental areas.     

Hatena arenicola gen. et sp. nov., a katablepharid undergoing probable plastid acquisition

Hatena arenicola gen. et sp. nov., an enigmatic flagellate of the katablepharids, is described. It shows ultrastructural affinities to the katablepharids, including large and small ejectisomes, cell covering, and a feeding apparatus. Although molecular phylogenies of the 18S ribosomal DNA support its classification into the katablepharids, the cell is characterized by a dorsiventrally compressed cell shape and a crawling motion, both of which are unusual within this group. The most distinctive feature of Hatena arenicola is that it harbors a Nephroselmis symbiont. This symbiosis is distinct from previously reported cases of ongoing symbiosis in that the symbiont plastid is selectively enlarged, while other structures such as the mitochondria, Golgi body, cytoskeleton, and endomembrane system are degraded; the host and symbiont have developed a morphological association, i.e., the eyespot of the symbiont is always at the cell apex of Hatena arenicola; and only one daughter cell inherits the symbiont during cell division, resulting in a symbiont-bearing green cell and a symbiont-lacking colorless cell. Interestingly, the colorless cells have a feeding apparatus that corresponds to the location of the eyespot in symbiont-bearing cells, and they are able to feed on prey cells. This indicates that the morphology of the host depends on the presence or absence of the symbiont. These observations suggest that Hatena arenicola has a unique "half-plant, half-predator" life cycle; one cell divides into an autotrophic cell possessing a symbiotic Nephroselmis species, and a symbiont-lacking colorless cell, which later develops a feeding apparatus de novo. The evolutionary implications of Hatena arenicola as an intermediate step in plastid acquisition are discussed in the context of other examples of ongoing endosymbioses in dinoflagellates.     

Symbiotic bacteria protect wasp larvae from fungal infestation

Symbiotic associations between different organisms are of great importance for evolutionary and ecological processes [1-4]. Bacteria are particularly valuable symbiotic partners owing to their huge diversity of biochemical pathways that may open entirely new ecological niches for higher organisms [1-3]. Here, we report on a unique association between a new Streptomyces species and a solitary hunting wasp, the European beewolf (Philanthus triangulum, Hymenoptera, Crabronidae). Beewolf females cultivate the Streptomyces bacteria in specialized antennal glands and apply them to the brood cell prior to oviposition. The bacteria are taken up by the larva and occur on the walls of the cocoon. Bioassays indicate that the streptomycetes protect the cocoon from fungal infestation and significantly enhance the survival probability of the larva, possibly by producing antibiotics. Behavioral observations strongly suggest a vertical transmission of the bacteria. Two congeneric beewolf species harbor closely related streptomycetes in their antennae, indicating that the association with protective bacteria is widespread among philanthine wasps and might play an important role in other insects as well. This is the first report on the cultivation of bacteria in insect antennae and the first case of a symbiosis involving bacteria of the important antibiotic-producing genus Streptomyces.     

Nitrite and nitrosyl compounds in food preservation.

Nitrite is consumed in the diet, through vegetables and drinking water. It is also added to meat products as a preservative. The potential risks of this practice are balanced against the unique protective effect against toxin-forming bacteria such as Clostridium botulinum. The chemistry of nitrite, and compounds derived from it, in food systems and bacterial cells are complex. It is known that the bactericidal species is not nitrite itself, but a compound or compounds derived from it during food preparation. Of a range of nitrosyl compounds tested, the anion of Roussin's black salt [Fe4S3(NO)7]- was the most inhibitory to C. sporogenes. This compound is active against both anaerobic and aerobic food-spoilage bacteria, while some other compounds are selective, indicating multiple sites of action. There are numerous possible targets for inhibition in the bacterial cells, including respiratory chains, iron-sulfur proteins and other metalloproteins, membranes and the genetic apparatus.     

Ammonia uptake and its effects on ionoregulation in the freshwater crayfish Pacifastacus leniusculus (Dana)

Exposure of adult crayfish Pacifastacus leniusculus to Artificial Freshwater (AFW) media containing 1.5 m and 0.15 mmol x l(-1) total ammonia [Tamm; 0.1 x acute lethal concentration (24 h LC50) and 0.01 x 24 h LC50] and adjusted to pH 6.5, pH 8.2 and pH 10.5 resulted in significant increases in haemolymph ammonia over a 24-h period. Ammonia accumulated most rapidly at pH 10.5. These media were chosen to expose animals to a range of different un-ionised ammonia (UIA) [NH3] and ionised ammonia [NH4+] concentrations. From comparisons of measured transepithelial potential differences (PDte) with calculated Nernst potentials (PDNH4+) for the known haemolymph-to-medium gradients of [NH4+], it was deduced that, in pH 8.2 and pH 6.5 AFW, NH4+ was not in thermodynamic equilibrium across the integument (presumably gill epithelium). In pH 10.5 AFW with 1.5 mmol x l(-1) Tamm (predominantly NH3), the accumulation of ammonia in the haemolymph was in the NH4+ form due to haemolymph pH regulation by the crayfish in this alkaline external medium. Measured net fluxes of ammonia (Jamm(net)) were inwardly directed and maximal when [NH3] was the main component externally, but were also significant at pH 8.2 with high [NH4+] ([NH4+]:[NH3] approximately 20:1). Haemolymph Na+ depletion was significant and, over the 24-h exposure period, most rapid in high [NH3] medium but [Cl-] was unaffected. However, paradoxically, sodium uptake (measured JNa(in) on immediate transfer to high Tamm medium) was not significantly inhibited when [NH3] was the predominant ammonia species. In 1.5 mmol x l(-1) Tamm (mainly [NH4+), VNa(in) (the active component of JNa(in)) was significantly inhibited, particularly at low external [Na+]. This inhibition could not be demonstrated as one of competition at an Na+/NH4+ apical gill exchange site. The resultant net efflux of sodium from the animal showed that the ability of the animals to balance sodium losses at low external [Na+] was severely affected. Longer exposure to pH 10.5 AFW with high [NH3] (12 h) resulted in significantly increased JNa(out), while not significantly affecting JNa(in). Analysis of urinary Na+ losses showed that, while urinary flow rate and water reabsorption was most likely unaffected by ammonia exposure, final urine [Na+] was significantly elevated. The resulting urinary Na+ loss accounted for 63% of the increased JNa(out) in high [NH3] medium.     

Biochemical and nucleic acid hybridisation studies on Brevibacterium linens and related strains

Twenty coryneform bacteria identified as Brevibacterium linens or related strains from different private and public collections were studied biochemically in respect to the composition of the cell walls and in respect to the nucleic acid hybridisation. Investigation of the cell walls revealed an identical meso-diaminopimelic acid containing directly cross-linked peptidoglycan type which is not amidated. The characteristic polymers of the polysaccharide moiety of the cell walls were found to be teichoic acids which belong to the poly(glycerolphosphate) and the poly(ribitolphosphate) type. Furthermore a novel mannitol containing teichoic acid is present which is tentatively characterized as poly(mannitolphosphate). Arabinoglactan and ribose as distinctive sugar components together with galactose and glucose in the cell walls of B. linens could not be detected in any strain. The biochemical findings lend support to the view that B. linens and related strains form a distinctive group which is clearly distinguished from all other coryneform bacteria. This is supported by DNA-23S/16S ribosomal ribonucleic acid reassociation studies. Deoxyribonucleic acid-deoxyribonucleic acid homology studies show the incoherency of B. linens which obviously comprises two species.     

Strict cospeciation of devescovinid flagellates and Bacteroidales ectosymbionts in the gut of dry-wood termites (Kalotermitidae)

The surface of many termite gut flagellates is colonized with a dense layer of bacteria, yet little is known about the evolutionary relationships of such ectosymbionts and their hosts. Here we investigated the molecular phylogenies of devescovinid flagellates (Devescovina spp.) and their symbionts from a wide range of dry-wood termites (Kalotermitidae). From species-pure flagellate suspensions isolated with micropipettes, we obtained SSU rRNA gene sequences of symbionts and host. Phylogenetic analysis showed that the Devescovina spp. present in many species of Kalotermitidae form a monophyletic group, which includes also the unique devescovinid flagellate Caduceia versatilis. All members of this group were consistently associated with a distinct lineage of Bacteroidales, whose location on the cell surface was confirmed by fluorescence in situ hybridization. The well-supported congruence of the phylogenies of devescovinids and their ectosymbionts documents a strict cospeciation. In contrast, the endosymbionts of the same flagellates ('Endomicrobia') were clearly polyphyletic and must have been acquired independently by horizontal transfer from other flagellate lineages. Also the Bacteroidales ectosymbionts of Oxymonas flagellates present in several Kalotermitidae belonged to several distantly related lines of descent, underscoring the general perception that the evolutionary history of flagellate-bacteria symbioses in the termite gut is complex.     

Organization and composition of the striated roots supporting the Golgi apparatus,the so-called parabasal apparatus,in parabasalid flagellates

Summary— In parabasalid flagellates, trichomonads and hypermastigids, the stack of cisternae of the Golgi apparatus are supported by striated roots attached to the basal bodies of flagella forming the so-called parabasal apparatus. Monoclonal antibodies raised for several trichomonad species, Monocercomonas, Trichomonas and Tetratrichomonas, label the parabasal fibre in immunofluorescence or immunogold staining and protein bands in immunoblotting. Several antibodies cross-react between trichomonad species, and one of them labels the homologous parabasal fibre in the hypermastigids: Trichonympha, Joenia, Pseudotrichonympha and Holomastigotoides. Considering the molecular mass range of the labelled proteins (100–135 kDa) and the lack of antibody cross-reactivity with the striated root proteins (centrin, assemblin, kinetodesmal protein, ciliary root proteins of epithelial ciliated cells) of other organisms, these proteins recognized by these antibodies seem to represent a new class of protein forming striated roots. The occurrence and significance of parabasal organization in eukaryogenesis is discussed.     

Paraphysomonas faveolata sp. nov. (Chrysophyceae), a fourth marine species with meshwork body-scales

Paraphysomonas faveolata sp. nov., the eighth described species of this genus of colourless chrysophycean flagellates, has heterokont flagellation, parabasal nucleus and silicified body-scales. It is the fourth known species with meshwork scales, these being of two types: flat “cobweb” scales and scales with a planar “honeycombed” extension arising from a “cobweb” base. The organism is compared with the other species of the genus and the case considered for placing forms with meshwork scales in a new genus, separate from forms with spined scales. This step is not taken and the taxonomic status of the genus is discussed in relation to the Ochromonadaceae and other genera of the Synuraceae.     

An ultrastructural comparison betweenSpermatozopsis andDunaliella (Chlorophyceae)

The ultrastructure of the type species of the genusDunaliella, D. salina, has been reinvestigated in an attempt to clarify the relationships betweenDunaliella andSpermatozopsis. Dunaliella salina differs in the following ultrastructural characters fromSpermatozopsis (as exemplified byS. similis Preisig etMelkonian): presence of a distinctive surface coat covering the plasmalemma; presence of a prominent pyrenoid (with pairs of thylakoids partially entering the pyrenoid matrix); dictyosomes parabasal; endoplasmic reticulum closely underlying the plasmalemma around most of the cell; contractile vacuoles absent; cell form ovoid to elongated and not spirally twisted; mitochondrial profiles near the flagellar apparatus. Differences in the ultrastructure of the flagellar apparatus: basal body angle more or less fixed; distal connecting fibre cross-striated; system II fibre (rhizoplast) present, associated with mitochondrial profile; system I fibre underlying two-stranded microtubular root; mating structure present. These ultrastructural differences justify distinction between the two taxa at generic level. The problematical status of freshwater species ofDunaliella is briefly discussed.     

Microbial contamination of cell cultures: a 2 years study.

Cell line contamination is a major drawback of main cell banks of the world and it has cost of losing important biological products or valuable research. The causative agents are different chemicals, invertebrates, bacteria, fungi, parasites, viral species and even other cell lines. In this retrospective study, cell lines from various species such as human, fish, insect, animals either offered or accessed through usual official accession in CGBRI were studied during 2 years (2002-2004) to detect their microbial contaminations and the causative organisms. Samples were taken for sterility test upon cell lines receipt and upon each cell line sub-culture. Samples were examined for bacterial (including mycoplasmas) and fungal contamination using conventional microbiological techniques. The study excluded parasites, viruses and other contaminating agents. This study revealed 39% of specimens were contaminated. The major contaminating agents were mycoplasmas (19%) followed by mixed infection (8%), fungi (8%) and bacteria (4%). Among various bacterial species (except mycoplasmas) Bacillus sp., Enterococcus sp. and Staphylococcus sp. are main bacterial agents and among various fungi Aspergillus sp. followed by Penicillium sp., Sepedonium sp. and Botrytis sp. were main fungal causative agents of CGBRI cell line contamination. Our study also delineates each cell line contamination rate and its causative agents. This is the first report of cell culture contamination from cell banks of Middle-East countries like Iran.