Characterization of new cristamonad species from kalotermitid termites including a novel genus, Runanympha.

Cristamonadea is a large class of parabasalian protists that reside in the hindguts of wood-feeding insects, where they play an essential role in the digestion of lignocellulose. This group of symbionts boasts an impressive array of complex morphological characteristics, many of which have evolved multiple times independently. However, their diversity is understudied and molecular data remain scarce. Here we describe seven new species of cristamonad symbionts from Comatermes, Calcaritermes, and Rugitermes termites from Peru and Ecuador. To classify these new species, we examined cells by light and scanning electron microscopy, sequenced the symbiont small subunit ribosomal RNA (rRNA) genes, and carried out barcoding of the mitochondrial large subunit rRNA gene of the hosts to confirm host identification. Based on these data, five of the symbionts characterized here represent new species within described genera: Devescovina sapara n. sp., Devescovina aymara n. sp., Macrotrichomonas ashaninka n. sp., Macrotrichomonas secoya n. sp., and Macrotrichomonas yanesha n. sp. Additionally, two symbionts with overall morphological characteristics similar to the poorly-studied and probably polyphyletic 'joeniid' Parabasalia are classified in a new genus Runanympha n. gen.: Runanympha illapa n. sp., and Runanympha pacha n. sp.

Spirotrichonymphea (Parabasalia) symbionts of the termite Paraneotermes simplicicornis.

The desert dampwood termite Paraneotermes simplicicornis harbors several species of obligately symbiotic protists that support its nutrition by fermenting lignocellulose. Among them are three morphotypes with the dexiotropic spiraling flagellar bands characteristic of Spirotrichonymphea (Parabasalia). The largest morphotype, characterized by an elongated cell apex with axial columella and internally positioned spiraling flagellar bands, was previously described as Spirotrichonympha polygyra. A smaller morphotype, with similarly internalized flagellar bands but a more rounded posterior without a protruding axostyle, was previously reported but not named. The smallest morphotype has surface flagellar bands and can attach to other protist cells by its apex. In this study, we combine light microscopy of live specimens and 18S rRNA gene sequencing of individually isolated cells to better understand the diversity of symbionts in P. simplicicornis. We found that S. polygyra branches distantly from true Spirotrichonympha, which are associated with Reticulitermes termites. Thus, we propose the new genus Cuppa to accommodate C. polygyra n. comb. (type species) and the similar but smaller morphotype Cuppa taenia n. sp. The undescribed smallest morphotype can be excluded from all previously described Spirotrichonymphea genera by molecular and behavioral evidence, so we propose Fraterculus simplicicornis n. gen., n. sp., to accommodate this organism.

Incomplete Co-cladogenesis Between Zootermopsis Termites and Their Associated Protists.

Coevolution is a major driver of speciation in many host-associated symbionts. In the termite-protist digestive symbiosis, the protists are vertically inherited by anal feeding among nest mates. Lower termites (all termite families except Termitidae) and their symbionts have broadly co-diversified over ~170 million yr. However, this inference is based mainly on the restricted distribution of certain protist genera to certain termite families. With the exception of one study, which demonstrated congruent phylogenies for the protist Pseudotrichonympha and its Rhinotermitidae hosts, coevolution in this symbiosis has not been investigated with molecular methods. Here we have characterized the hindgut symbiotic protists (Phylum Parabasalia) across the genus Zootermopsis (Archotermopsidae) using single cell isolation, molecular phylogenetics, and high-throughput amplicon sequencing. We report that the deepest divergence in the Zootermopsis phylogeny (Zootermopsis laticeps [Banks; Isoptera: Termopsidae]) corresponds with a divergence in three of the hindgut protist species. However, the crown Zootermopsis taxa (Zootermopsis angusticollis [Hagen; Isoptera: Termopsidae], Z. nevadensis nevadensis [Hagen; Isoptera: Termopsidae], and Z. nevadensis nuttingi [Haverty & Thorne; Isoptera: Termopsidae]) share the same protist species, with no evidence of co-speciation under our methods. We interpret this pattern as incomplete co-cladogenesis, though the possibility of symbiont exchange cannot be entirely ruled out. This is the first molecular evidence that identical communities of termite-associated protist species can inhabit multiple distinct host species.

Host-Symbiont Cospeciation of Termite-Gut Cellulolytic Protists of the Genera Teranympha and Eucomonympha and their Treponema Endosymbionts.

Cellulolytic flagellated protists inhabit the hindgut of termites. They are unique and essential to termites and related wood-feeding cockroaches, enabling host feeding on cellulosic matter. Protists of two genera in the family Teranymphidae (phylum Parabasalia), Eucomonympha and Teranympha, are phylogenetically closely related and harbor intracellular endosymbiotic bacteria from the genus Treponema. In order to obtain a clearer understanding of the evolutionary history of this triplex symbiotic relationship, the molecular phylogenies of the three symbiotic partners, the Teranymphidae protists, their Treponema endosymbionts, and their host termites, were inferred and compared. Strong congruence was observed in the tree topologies of all interacting partners, implying their cospeciating relationships. In contrast, the coevolutionary relationship between the Eucomonympha protists and their endosymbionts was more complex, and evidence of incongruence against cospeciating relationships suggested frequent host switches of the endosymbionts, possibly because multiple Eucomonympha species are present in the same gut community. Similarities in the 16S rRNA and gyrB gene sequences of the endosymbionts were higher among Teranympha spp. (>99.25% and >97.2%, respectively), whereas those between Teranympha and Eucomonympha were lower (<97.1% and <91.9%, respectively). In addition, the endosymbionts of Teranympha spp. formed a phylogenetic clade distinct from those of Eucomonympha spp. Therefore, the endosymbiont species of Teranympha spp., designated here as "Candidatus Treponema teratonymphae", needs to be classified as a species distinct from the endosymbiont species of Eucomonympha spp.

Pseudotrichonympha leei, Pseudotrichonympha lifesoni, and Pseudotrichonympha pearti, new species of parabasalian flagellates and the description of a rotating subcellular structure.

Pseudotrichonympha is a large and structurally complex genus of parabasalian protists that play a key role in the digestion of lignocellulose in the termite hindgut. Like many termite symbionts, it has a conspicuous body plan that makes genus-level identification relatively easy, but species-level diversity of Pseudotrichonympha is understudied. Molecular surveys have suggested the diversity is much greater than the current number of described species, and that many "species" described in multiple hosts are in fact different, but gene sequences from formally described species remain a rarity. Here we describe three new species from Coptotermes and Prorhinotermes hosts, including small subunit ribosomal RNA (SSU rRNA) sequences from single cells. Based on host identification by morphology and DNA barcoding, as well as the morphology and phylogenetic position of each symbiont, all three represent new Pseudotrichonympha species: P. leei, P. lifesoni, and P. pearti. Pseudotrichonympha leei and P. lifesoni, both from Coptotermes, are closely related to other Coptotermes symbionts including the type species, P. hertwigi. Pseudotrichonympha pearti is the outlier of the trio, more distantly related to P. leei and P. lifesoni than they are to one another, and contains unique features, including an unusual rotating intracellular structure of unknown function.

Effects of lignins as diet components on the physiological activities of a lower termite, Coptotermes formosanus Shiraki.

We investigated the effects of lignins as diet components on the physiological activities of a lower termite, Coptotermes formosanus Shiraki. Artificial diets composed of polysaccharides with and without purified lignins (milled-wood lignins) from Japanese cedar (softwood), Japanese beech (hardwood), and rice (grass), were fed to C. formosanus workers. The survival and body mass of the workers as well as the presence of three symbiotic protists in the hindguts of the workers were then periodically examined. The survival rates of workers fed on diets containing lignins were, regardless of the lignocellulose diet sources, significantly higher than those of workers fed on only polysaccharides. In addition, it was clearly observed that all the tested lignins have positive effects on the maintenance of two major protists in the hindguts of C. formosanus workers, i.e., Pseudotrichonympha grassii and Holomastigotoides hartmanni. Overall, our data suggest that the presence of lignin is crucial to maintaining the physiological activities of C. formosanus workers during their lignocellulose decomposition. Our data also suggested that some components, possibly minerals and/or non-structural carbohydrates, in grass lignocellulose negatively affect the survival of C. formosanus workers as well as the present rate of the symbiotic protists in their hindguts.

Genome Evolution and Nitrogen Fixation in Bacterial Ectosymbionts of a Protist Inhabiting Wood-Feeding Cockroaches.

UNLABELLED: By combining genomics and isotope imaging analysis using high-resolution secondary ion mass spectrometry (NanoSIMS), we examined the function and evolution of Bacteroidales ectosymbionts of the protist Barbulanympha from the hindguts of the wood-eating cockroach Cryptocercus punctulatus In particular, we investigated the structure of ectosymbiont genomes, which, in contrast to those of endosymbionts, has been little studied to date, and tested the hypothesis that these ectosymbionts fix nitrogen. Unlike with most obligate endosymbionts, genome reduction has not played a major role in the evolution of the Barbulanympha ectosymbionts. Instead, interaction with the external environment has remained important for this symbiont as genes for synthesis of transporters, outer membrane proteins, lipopolysaccharides, and lipoproteins have been retained. The ectosymbiont genome carried two complete operons for nitrogen fixation, a urea transporter, and a urease, indicating the availability of nitrogen as a driving force behind the symbiosis. NanoSIMS analysis of C. punctulatus hindgut symbionts exposed in vivo to (15)N2 supports the hypothesis that Barbulanympha ectosymbionts are capable of nitrogen fixation. This genomic and in vivo functional investigation of protist ectosymbionts highlights the diversity of evolutionary forces and trajectories that shape symbiotic interactions. IMPORTANCE: The ecological and evolutionary importance of symbioses is increasingly clear, but the overall diversity of symbiotic interactions remains poorly explored. In this study, we investigated the evolution and nitrogen fixation capabilities of ectosymbionts attached to the protist Barbulanympha from the hindgut of the wood-eating cockroach Cryptocercus punctulatus In addressing genome evolution of protist ectosymbionts, our data suggest that the ecological pressures influencing the evolution of extracellular symbionts clearly differ from intracellular symbionts and organelles. Using NanoSIMS analysis, we also obtained direct imaging evidence of a specific hindgut microbe playing a role in nitrogen fixation. These results demonstrate the power of combining NanoSIMS and genomics tools for investigating the biology of uncultivable microbes. This investigation paves the way for a more precise understanding of microbial interactions in the hindguts of wood-eating insects and further exploration of the diversity and ecological significance of symbiosis between microbes.

Intracloacally passaged low-virulent Histomonas meleagridis protects turkeys from histomonosis.

Histomonosis (blackhead disease or infectious enterohepatitis) caused by the extracellular protozoon parasite Histomonas meleagridis is an important disease of turkeys and a threat to the poultry industry. Due to recent legislation on drug restrictions, research to find new alternatives is an urgent matter in the battle against histomonosis. In the present study, intracloacal inoculation of a low-virulent H. meleagridis strain isolated after serial passages in turkeys clearly demonstrated a reduction of virulence and hence its effectiveness as a vaccine against histomonosis. The low-virulent isolate has been evaluated in a comparative experimental infection study. No mortality nor predominant caecal or liver lesions could be observed in the groups inoculated with 10(3), 10(4) or 10(5) histomonads per bird. Only dilated caeca with a yellow and foamy content could be noticed. Groups inoculated with similar doses of a virulent strain displayed a dose-related pathology and mortality up to 100%. The protective capacity of the strain with reduced virulence could be demonstrated as none of the birds cloacally inoculated with 10(3), 10(4) or 10(5) histomonads died upon challenge with 10(5)H. meleagridis of the virulent strain. Hereby, 71% of the challenge control group died. Interestingly, no or only very minor pathological lesions in the caeca and liver could be detected after challenge of the birds inoculated with the passaged histomonads. In conclusion, cloacal inoculation of the low-virulent strain obtained after serial backpassages was able to induce protection of turkeys against challenge with a virulent H. meleagridis strain.

Cthulhu Macrofasciculumque n. g., n. sp. and Cthylla Microfasciculumque n. g., n. sp., a newly identified lineage of parabasalian termite symbionts.

The parabasalian symbionts of lower termite hindgut communities are well-known for their large size and structural complexity. The most complex forms evolved multiple times independently from smaller and simpler flagellates, but we know little of the diversity of these small flagellates or their phylogenetic relationships to more complex lineages. To understand the true diversity of Parabasalia and how their unique cellular complexity arose, more data from smaller and simpler flagellates are needed. Here, we describe two new genera of small-to-intermediate size and complexity, represented by the type species Cthulhu macrofasciculumque and Cthylla microfasciculumque from Prorhinotermes simplex and Reticulitermes virginicus, respectively (both hosts confirmed by DNA barcoding). Both genera have a single anterior nucleus embeded in a robust protruding axostyle, and an anterior bundle flagella (and likely a single posterior flagellum) that emerge slightly subanteriorly and have a distinctive beat pattern. Cthulhu is relatively large and has a distinctive bundle of over 20 flagella whereas Cthylla is smaller, has only 5 anterior flagella and closely resembles several other parababsalian genera. Molecular phylogenies based on small subunit ribosomal RNA (SSU rRNA) show both genera are related to previously unidentified environmental sequences from other termites (possibly from members of the Tricercomitidae), which all branch as sisters to the Hexamastigitae. Altogether, Cthulhu likely represents another independent origin of relatively high cellular complexity within parabasalia, and points to the need for molecular characterization of other key taxa, such as Tricercomitus.

Molecular characterization of parabasalian symbionts Coronympha clevelandii and Trichonympha subquasilla from the Hawaiian lowland tree termite Incisitermes immigrans.

An important and undervalued challenge in characterizing symbiotic protists is the accurate identification of their host species. Here, we use DNA barcoding to resolve one confusing case involving parabasalian symbionts in the hindgut of the Hawaiian lowland tree termite, Incisitermes immigrans, which is host to several parabasalians, including the type species for the genus Coronympha, C. clevelandii. We collected I. immigrans from its type locality (Hawaii), confirmed its identity by DNA barcoding, and characterized the phylogenetic position of two symbionts, C. clevelandii and Trichonympha subquasilla. These data show that previous molecular surveys of "I. immigrans" are, in fact, mainly derived from the Caribbean termite I. schwarzi, and perhaps also another related species. These results emphasize the need for host barcoding, clarify the relationship between morphologically distinct Coronympha species, and also suggest some interesting distribution patterns of nonendemic termite species and their symbionts.

Symbiosis, morphology, and phylogeny of Hoplonymphidae (Parabasalia) of the wood-feeding roach Cryptocercus punctulatus.

Anaerobic cellulolytic flagellate protists of the hindguts of lower termites and the wood-feeding cockroach Cryptocercus are essential to their host's ability to digest lignocellulose. Many have bacteria associated with their surfaces and within cytoplasmic vesicles-likely important symbioses as suggested by molecular and other data. Some of the most striking examples of these symbioses are in the parabasalid family Hoplonymphidae, but little or no data exist on the structural aspects of their symbioses, their relationships with bacteria through different life-cycle stages, or their diversity and phylogenetic relationships in Cryptocercus. We investigated these areas in the hoplonymphid genera Barbulanympha and Urinympha from Cryptocercus punctulatus using light and electron microscopy, and analysis of small subunit rRNA. Microscopy reveals variation in density of bacterial surface symbionts related to life-cycle stage, a glyococalyx possibly important in bacterial adhesion and/or metabolite exchange, and putative viruses associated with bacterial surface symbionts. Patterning of surface bacteria suggests protists emerging from the resistant (dormant) stage are colonized by a small population of bacterial cells, which then divide to cover their surface. Additionally, cytoplasmic protrusions from the protist are covered by bacteria. Phylogenetic analysis rejects the monophyly of Hoplonymphidae, suggesting multiple origins or losses of these bacterial symbioses.

Identification and localization of the multiple bacterial symbionts of the termite gut flagellate Joenia annectens.

The hindgut of wood-feeding lower termites is densely colonized by a multitude of symbiotic micro-organisms. While it is well established that the eukaryotic flagellates play a major role in the degradation of lignocellulose, much less is known about the identity and function of the prokaryotic symbionts associated with the flagellates. Our ultrastructural investigations of the gut flagellate Joenia annectens (from the termite Kalotermes flavicollis) revealed a dense colonization of this flagellate by diverse ecto- and endosymbiotic bacteria. Phylogenetic analysis of the small-subunit rRNA gene sequences combined with fluorescence in situ hybridization allowed us to identify and localize the different morphotypes. Furthermore, we could show that K. flavicollis harbours two phylotypes of J. annectens that could be distinguished not only by their small-subunit rRNA gene sequences, but also by differences in their assemblages of bacterial symbionts. Each of the flagellate populations hosted phylogenetically distinct ectosymbionts from the phylum Bacteroidetes, one of them closely related to the ectosymbionts of other termite gut flagellates. A single phylotype of 'Endomicrobia' was consistently associated with only one of the host phylotypes, although not all individuals were colonized, corroborating that 'Endomicrobia' symbionts do not always cospeciate with their host lineages. Flagellates from both populations were loosely associated with a single phylotype of Spirochaetales attached to their cell surface in varying abundance. Current evidence for the involvement of Bacteroidales and 'Endomicrobia' symbionts in the nitrogen metabolism of the host flagellate is discussed.