Renate Radek

“Endomicrobia”: Cytoplasmic Symbionts of Termite Gut Protozoa Form a Separate Phylum of Prokaryotes

ABSTRACT Lignocellulose digestion by wood-feeding termites depends on the mutualistic interaction of unusual, flagellate protists located in their hindgut. Most of the flagellates harbor numerous prokaryotic endosymbionts of so-far-unknown identity and function. Using a full-cycle molecular approach, we show here that the endosymbionts of the larger gut flagellates of Reticulitermes santonensis belong to the so-called termite group 1 (TG-1) bacteria, a group of clones previously obtained exclusively from gut homogenates of Reticulitermes speratus that are only distantly related to other bacteria and are considered a novel bacterial phylum based on their 16S rRNA gene sequences. Fluorescence in situ hybridization with specifically designed oligonucleotide probes confirmed that TG-1 bacteria are indeed located within the flagellate cells and demonstrated that Trichonympha agilis (Hypermastigida) and Pyrsonympha vertens (Oxymonadida) harbor phylogenetically distinct populations of symbionts (<95% sequence similarity). Transmission electron microscopy revealed that the symbionts are small, spindle-shaped cells (0.6 μm in length and 0.3 μm in diameter) surrounded by two membranes and located within the cytoplasm of their hosts. The symbionts of the two flagellates are described as candidate species in the candidate genus “Endomicrobium.” Moreover, we provide evidence that the members of the TG-1 phylum, for which we propose the candidate name “Endomicrobia,” are phylogenetically extremely diverse and are present in and also restricted to the guts of all lower termites and wood-feeding cockroaches of the genus Cryptocercus, the only insects that are in an exclusive, obligately mutualistic association with such unique cellulose-fermenting protists.

Identification of the ectosymbiotic bacteria of Mixotricha paradoxa involved in movement symbiosis

Mixotricha paradoxa, a trichomonad from the hindgut of the Australian termite Mastotermes darwiniensis Froggatt, is a rare example of a movement symbiosis between eukaryotic and prokaryotic microorganisms. The surface of Mixotricha paradoxa is covered with spirochaetes and a rod-shaped bacterium. The four flagella at the anterior end seem only to alter the direction of movement, while the ectosymbiotic spirochaetes propel the flagellate cells. Based on a 16S rDNA sequence analysis after a semi-specific PCR, and subsequent fluorescence in situ hybridization applying helper oligonucleotides and a denaturing step of the 16S rRNA, three different spirochaete clones could be clearly identified on the surface of the protozoal cells. They belonged to the Treponema cluster. The rod shaped bacterium showed highest 16S rDNA sequence similarity to species related to Bacteroides. Due to its low phylogenetic relationship to its nearest relatives in the database, it should represent a so far undescribed species.

Symbiotic Protozoa of Termites

Lower termites and the roachCryptocercusharbour in their hindgut a dense fauna of flagellate protozoa mixed with other microorganisms, such as bacteria, archaebacteria and yeasts. The termite hindgut and its symbionts formamicroaerophilic ecosystemthat acts in thedigestionofplantmaterial ingested by these insects. This chapter presents the diversity of the flagellate protozoa, their cytological features and phylogeny (Part 1) and several aspects of the biology and adaptations of these eukaryotes to this particular ecosystem (Part 2).

Spirochaeta coccoides sp. nov., a Novel Coccoid Spirochete from the Hindgut of the Termite Neotermes castaneus

ABSTRACT A novel spirochete strain, SPN1, was isolated from the hindgut contents of the termite Neotermes castaneus. The highest similarities (about 90%) of the strain SPN1 16S rRNA gene sequence are with spirochetes belonging to the genus Spirochaeta, and thus, the isolate could not be assigned to the so-called termite clusters of the treponemes or to a known species of the genus Spirochaeta. Therefore, it represents a novel species, which was named Spirochaeta coccoides. In contrast to all other known validly described spirochete species, strain SPN1 shows a coccoid morphology and is immotile. The isolated strain is obligately anaerobic and ferments different mono-, di-, and oligosaccharides by forming formate, acetate, and ethanol as the main fermentation end products. Furthermore, strain SPN1 is able to grow anaerobically with yeast extract as the sole carbon and energy source. The fastest growth was obtained at 30°C, the temperature at which the termites were also grown. The cells possess different enzymatic activities that are involved in the degradation of lignocellulose in the termite hindgut, such as β-d-glucosidase, α-l-arabinosidase, and β-d-xylosidase. Therefore, they may play an important role in the digestion of breakdown products from cellulose and hemicellulose in the termite gut.

How oxymonads lost their groove: An ultrastructural comparison of Monocercomonoides and excavate taxa

Abstract Despite being amongst the more familiar groups of heterotrophic flagellates, the evolutionary affinities of oxymonads remain poorly understood. A re-interpretation of the cytoskeleton of the oxymonad Monocercomonoides hausmanni suggests that this organism has a similar ultrastructural organisation to members of the informal assemblage ‘excavate taxa’. The preaxostyle, ‘R1’ root, and ‘R2’ root of M. hausmanni are proposed to be homologous to the right, left, and anterior roots respectively of excavate taxa. The ‘paracrystalline’ portion of the preaxostyle, previously treated as unique to oxymonads, is proposed to be homologous to the I fibre of excavate taxa. Other non-microtubular fibres are identified that have both positional and substructural similarity to the distinctive B and C fibres of excavate taxa. A homologue to the ‘singlet root’, otherwise distinctive for excavate taxa, is also proposed. The preaxostyle and C fibre homologue in Monocercomonoides are most similar to the homologous structures in Trimastix, suggesting a particularly close relationship. This supports and extends recent molecular phylogenetic findings that Trimastix and oxymonads form a clade. We conclude that oxymonads have an excavate ancestry, and that the ‘excavate taxa’ sensu stricto form a paraphyletic assemblage.

Light and electron microscopic study of the bacterial adhesion to termite flagellates applying lectin cytochemistry

SummaryMany of the flagellates inhabiting the hindgut of lower termites are associated with ectobiotic, rod-like bacteria or spirochetes. Different types of attachment sites are present. Electron dense material underlies, e.g., the plasma membrane ofJoenia annectens at the contact site, whereas other attachment sites do not show any visible specializations. The host cells glycocalyx may, however, be reduced at the attachment sites as it is the case inDevescovina glabra. The thick glycocalyx ofStephanonympha nelumbium is not changed at the sites where bacterial rods attach, but spirochetes penetrate to a certain extent. Bacteria which colonize the extracellular surface structures ofMicrorhopalodina multinucleata express their own glycocalyx to mediate a contact. In this study we focussed on the examination of one common mode of interaction between bacteria and their host cells, i.e., adhesion via lectins and sugars. The sugar composition was analysed by light and electron microscopic labelling experiments using the lectins Con A, WGA and SBA. In general, only the posterior body surface ofJoenia which is colonized with bacteria is labelled. The demonstrated sugars are found in fibrous glycocalyx portions surrounding the attachment sites of the bacteria. Such glycocalyx fibres in combination with the electron dense material supporting the attachment sites seem to be the prerequisites for bacterial attachment. InD. glabra, however, a role for sugars in mediating the attachment could not be demonstrated. Removal of the ectobiotes using antibiotics revealed that the specialized contact sites ofJoenia are present in the absence of bacteria and thus possibly serve to attract bacteria. Nothing, however, remains of the former attachment sites in bacteria-freeDevescovina cells. Attachment sites in this case could be induced by bacterial contact. There is not one general mechanism for bacterial attachment to termite flagellates; rather, adhesion seems to follow different strategies.

Monocercomonoides termitis n. sp., an Oxymonad from the Lower Termite Kalotermes sinaicus

Summary: The characteristic features of the new termite flagellate Monocercomonoides termitis n. Spa are: clavate body measuring 7 to 9.6 (8.3) µm in length and 3.5 to 6.1 (4.9) µm in width; 4 anterior flagella of about twice the body length, recurrent flagellum is slightly longer and attached to the cell body for a short distance; large, spherical nucleus (2.9 µm); protruding axostyle; pelta. As it is typical of the order Oxymonadida the basal bodies of the four flagella are arranged in two pairs which are connected by a bowl-like, paracrystalline preaxostyle. Golgi apparatus and mitochondria or hydrogenosomes are missing. Since neither a permanent nor a transitory holdfast organelle is present the described specimen is a member of the family Polymastiqidae.

Bacterial adhesion to different termite flagellates: Ultrastructural and functional evidence for distinct molecular attachment modes

SummaryThe attachment modes of rodlike ectobiotic bacteria to the surface of two different termite flagellates were studied.Devescovina glabra was covered by laterally attached bacteria. Treatment with chemicals that disturb hydrophobic interactions and solubilize proteins removed the ectobionts. Freeze-fracture and freeze-etching electron microscopy revealed rows of intramembrane particles that occurred exclusively along the attachment sites. The adhering Gram-negative bacteria possessed an S-layer (surface layer) composed of globular protein particles. The S-layer could be removed by protein-solubilizing chemicals, e.g., urea, as shown by ultrathin-section electron microscopy. Therefore, it seems plausible that the attachment was mediated by hydrophobic interactions between the flagellates plasma membrane and the S-layer of the bacteria. The bacteria of the second flagellate,Joenia annectens, adhered by their tips. The attachment was extremely strong. Chemicals disturbing ionic or hydrophobic bindings or solubilizing proteins did not detach the ectobionts. Globular intramembrane protein particles were preferentially found in a ringlike array at the external fracture face of the flagellates contact sites.

Spirotrichonympha minor n. sp., a new hypermastigote termite flagellate

Summary Spirotrichonympha minor n. sp. is an undescribed hypermastigote flagellate from the lower termite Kalotermes sinaicus . It was examined light microscopically in live and protargol stained preparations and by scanning and transmission electron microscopy. S. minors main characteristics are: body ovoid to pear-shaped (8–31 × 5–22 μm); two to eight rows of flagella which are arranged in a righthanded spiral of about one turn, the flagellar rows end near the posterior end of the body; a rounded or elongate nucleus (4×3.2 μm) lying close to the anterior end of the cell; rounded to elongate parabasal bodies which follow the course of the flagellar bands; several anterior branches of the axostyle coalesce into a thin trunk posterior to the nucleus, rarely protruding at the posterior cell pole; and endocytobiotic methanogenic bacteria. Ultrastructurally visible features are: tapering flagella part of which are equipped with a brush-like glycocalyx; anterior basal bodies of the flagellar bands lying perpendicular to a tube-like structure of the rostrum; and no permanent atractophores. The reasons to place Spirotrichonympha minor n. sp. in the genus Spirotrichonympha and to create a new species are discussed.

Placojoenia sinaica n. g., n. sp., a symbiotic flagellate from the termite Kalotermes sinaicus

Summary The hypermastigote flagellate Placojoenia sinaica n. g., n. sp., symbiot of the termite Kalotermes sinaicus , has been examined by light and electron microscopy. The new genus belongs to the family Joeniidae and possesses the following characteristics: horseshoe-shaped tuft of flagella; parabasal bodies wound around the axostyle and some additional parabasal bodies scattered in the cytoplasm; cytoplasmic plates. Placojoenia sinaica has a clavate body (200 × 104 μm) and a posteriorly protruding axostyle. The microtubular axostyle folds up to a capitulum anteriorly which encloses the nucleus, numerous hydrogenosomes, and spiral-shaped bacteria. The nuclear envelope forms branching, tubular extensions whose meshwork fills a wide perinuclear region. The cytoplasmic plates are aggregates of glycogen surrounded by cisterns of endoplasmic reticulum. Rod-shaped bacteria located in the plates often contain virus-like particles. Periodically striated filaments form the parabasal plates; they emerge near a structure that supports the flagellar tuft and whose shape is roughly congruent to that of the tuft. The basal bodies of the flagella are extraordinarily long (2.8 to 3.9 μm).