Exclusive Gut Flagellates of Serritermitidae Suggest a Major Transfaunation Event in Lower Termites: Description of Heliconympha glossotermitis gen. nov. spec. nov.

The guts of lower termites are inhabited by host-specific consortia of cellulose-digesting flagellate protists. In this first investigation of the symbionts of the family Serritermitidae, we found that Glossotermes oculatus and Serritermes serrifer each harbor similar parabasalid morphotypes: large Pseudotrichonympha-like cells, medium-sized Leptospironympha-like cells with spiraled bands of flagella, and small Hexamastix-like cells; oxymonadid flagellates were absent. Despite their morphological resemblance to Pseudotrichonympha and Leptospironympha, a SSU rRNA-based phylogenetic analysis identified the two larger, trichonymphid flagellates as deep-branching sister groups of Teranymphidae, with Leptospironympha sp. (the only spirotrichosomid with sequence data) in a moderately supported basal position. Only the Hexamastix-like flagellates are closely related to trichomonadid flagellates from Rhinotermitidae. The presence of two deep-branching lineages of trichonymphid flagellates in Serritermitidae and the absence of all taxa characteristic of the ancestral rhinotermitids underscores that the flagellate assemblages in the hindguts of lower termites were shaped not only by a progressive loss of flagellates during vertical inheritance but also by occasional transfaunation events, where flagellates were transferred horizontally between members of different termite families. In addition to the molecular phylogenetic analyses, we present a detailed morphological characterization of the new spirotrichosomid genus Heliconympha using light and electron microscopy.

New Species of Spirotrichonympha from Reticulitermes and the Relationships Among Genera in Spirotrichonymphea (Parabasalia).

Spirotrichonymphea is a class of hypermastigote parabasalids defined by their spiral rows of many flagella. They are obligate hindgut symbionts of lower termites. Despite more than 100 yr of morphological and ultrastructural study, the group remains poorly characterised by molecular data and the phylogenetic positions and taxonomic validity of most genera remain in question. The genus Spirotrichonympha has been reported to inhabit several termite genera, including Reticulitermes, Coptotermes, and Hodotermopsis. The type species for this genus, Spirotrichonympha flagellata, was described from Reticulitermes lucifugus but no molecular data are yet available for this species. In this study, three new Spirotrichonympha species are described from three species of Reticulitermes. Their molecular phylogenetic position indicates that the genus is not monophyletic, as Spirotrichonympha species from Coptotermes, Paraneotermes, and Hodotermopsis branch separately. In contrast, the genus Holomastigotoides is monophyletic, as demonstrated using new sequences from Holomastigotoides species. The presence of Holomastigotoides in Prorhinotermes and the distinct phylogenetic positions of Spirotrichonympha from Reticulitermes and Coptotermes are consistent with a previously proposed symbiont fauna replacement in the ancestor of Reticulitermes.

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.

Light and transmission electron microscopic studies on the encystation of Histomonas meleagridis.

The study deals with the pleomorphic zooflagellate Histomonas meleagridis, which was cultivated under different stress conditions to induce a possible encystation. In the present paper, the morphological changes were analyzed by light and electron microscopy. The determination of the proliferation under different adverse conditions led to conclusions on the tenacity of the flagellate. H. meleagridis parasitizes in the intestinal tract of galliform birds and may cause enormous losses in poultry farming. For the development of new therapy approaches, clarification of the transmission pathways will be helpful. Different clonal cultures of H. meleagridis established by micromanipulation and exposed to media lacking different ingredients, inappropriate temperatures, and/or distinct reagents were investigated. Lowering of temperature was proven to have adverse effects on the survival of H. meleagridis. The flagellate could not survive in a frozen medium, and survival in a temperature of 4 degrees C lasted no longer than 23 h. An addition of sodium chloride induced an increased proliferation; pH values between 2 and 8 set limits for the survival of the parasite in different ways. H. meleagridis was able to survive under high acidic conditions for only 1 h. The major amount of cells, which could be discovered in the controls, measured 8-12 microm appeared amoebic (stage 1) and were filled with enclosures of rice starch. A rounding of most cells was noted 4 h at 4 degrees C after incubation in minimal essential medium in the absence of rice starch and fetal calf serum. A higher osmolarity of the medium, which was initiated by the addition of sodium chloride or magnesium chloride, did not induce an encystation process. After addition of hypochlorite base and cultivating at pH values between 7 and 8, spherical stages without a flagellum were formed (stage 2) measuring about 8-12 microm in diameter. Their interior consisted of a central and a peripheral region when studied by transmission electron microscopy. This aspect was due to the location of the glycogen granules. The central zone was described as totally filled with the carbohydrates, which made totally invisible the other organelles. The solidity of the amorphous layer below the cell membrane seemed to hinder the invasion of the glycogen granules. The amorphous layer below the cell membrane made it apparently possible that the cell might survive under adverse conditions-at least for a short time. This special structure might enable H. meleagridis to proceed a fast transmission and to infect many birds in a rather short time, which was shown in the past by several studies. Double-membraned cells, which were guessed to be cyst-like structures of the parasite, were also detected (stage 3). The size of these cells, however, was much smaller than that of the amoebic stages or the above-described spherical forms of H. meleagridis. Furthermore, the small cells were characterized by other granula structures. These findings might be interpreted that the small stages are possibly long-term (true) cysts and that the spherical stages with the amorphous layer beneath the cell membrane might be short-term cysts. Both, however, should be able to survive situations outside of a body and thus might be transmitted from feces to another animal.

Critical taxonomic revision of Parabasalids with description of one new genus and three new species.

We propose a new classification of Parabasalia which is congruent with both ultrastructural and molecular-phylogenetic studies. We identify six main parabasalid lineages and give them the rank of class: Hypotrichomonadea, Trichomonadea, Tritrichomonadea, Cristamonadea, Trichonymphea, and Spirotrichonymphea. Trichomonadea is characterized by a single mastigont and by the absence of both a comb-like structure and an infrakinetosomal body. Most representatives also possess a lamelliform undulating membrane. Trichomonadea is divided into two monophyletic orders, Trichomonadida (family Trichomonadidae; with a B-type costa) and Honigbergiellida (families Honigbergiellidae, Hexamastigidae and Tricercomitidae; without a costa). The class Tritrichomonadea, with a single order Tritrichomonadida, is ancestrally characterized by a single mastigont with four flagella, and both a comb-like structure and an infrakinetosomal body. The morphologically most complex representatives (family Tritrichomonadidae) possess in addition a rail-type undulating membrane, an A-type costa, and a suprakinetosomal body. These last three characters are absent in families Monocercomonadidae and Simplicimonadidae. The remaining tritrichomonadids, Dientamoebidae, have undergone reductive evolution. Cristamonads (Cristamonadea) are morphologically derived from tritrichomonads. Because we are unable to determine morphologically homogenous monophyletic lineages within cristamonads, we classify all cristamonads into a single family, Lophomonadidae. Hypotrichomonadea, comprising the genera Trichomitus and Hypotrichomonas, resembles Tritrichomonadea by an A-type costa, and by the presence of a comb-like structure in the mastigont. However, they do not possess an infrakinetosomal body, and are not specifically related to Tritrichomonadea in molecular-phylogenetic analyses. Moreover, unlike Tritrichomonadea, Hypotrichomonadea possesses a lamelliform undulating membrane. The remaining parabasalids are of complex morphology and belong to the classes Trichonymphea and Spirotrichonymphea. A new parabasalid genus, Simplicimonas (Tritrichomonadea), and three new species, Tetratrichomonas undula, Hexamastix coercens and Simplicimonas similis, are described.

Phylogenetic position and morphology of spirotrichosomidae (parabasalia): new evidence from Leptospironympha of Cryptocercus punctulatus.

Parabasalia are a large, diverse clade of anaerobic flagellates, many of which inhabit the guts of wood-feeding insects. Because most are uncultivable, molecular data representing the true diversity of Parabasalia only became possible with the application of single-cell techniques, but in the last decade molecular data have accumulated rapidly. Within the Trichonymphida, the most diverse lineage of hypermastigote parabasalids, molecular data are now available from five of the six families, however, one family, the Spirotrichosomidae, has not been sampled at the molecular level, and is very little studied with electron microscopy. Here we examine a representative of Spirotrichosomidae--Leptospironympha of the wood-feeding cockroach Cryptocercus punctulatus--with scanning and transmission electron microscopy, and analyze its small subunit rRNA gene to infer its phylogenetic position. Phylogenetic analyses place Leptospironympha as sister to a clade comprising Eucomonymphidae and Teranymphidae with moderate support. Examination with scanning and transmission electron microscopy reveals new classes of previously undetected symbiotic surface bacteria, a glycocalyx, granular particles on flagella, and putative phagocytosed bacteria. The range of flagellar patterns in Spirotrichosomidae is quite wide, and the possibility that some members may be more closely related to Eucomonymphidae or Teranymphidae is addressed.