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.

Molecular Evidence for the Polyphyly of Macrotrichomonas (Parabasalia: Cristamonadea) and a Proposal for Macrotrichomonoides n. gen.

Macrotrichomonas (Cristamonadea: Parabasalia) is an anaerobic, amitochondriate flagellate symbiont of termite hindguts. It is noteworthy for being large but not structurally complex compared with other large parabasalians, and for retaining a structure similar in appearance to the undulating membrane (UM) of small flagellates closely related to cristamonads, e.g. Tritrichomonas. Here, we have characterised the SSU rDNA from two species described as Macrotrichomonas: M. restis Kirby 1942 from Neotermes jouteli and M. lighti Connell 1932 from Paraneotermes simplicicornis. These species do not form a clade: M. lighti branches with previously characterised Macrotrichomonas sequences from Glyptotermes, while M. restis branches with the genus Metadevescovina. We examined the M. restis UM by light microscopy, scanning electron microscopy, and transmission electron microscopy, and we find common characteristics with the proximal portion of the robust recurrent flagellum of devescovinids. Altogether, we show the genus Macrotrichomonas to be polyphyletic and propose transferring M. restis to a new genus, Macrotrichomonoides. We also hypothesise that the macrotrichomonad body plan represents the ancestral state of cristamonads, from which other major forms evolved.

The phylogenetic position of Kofoidia loriculata (Parabasalia) and its implications for the evolution of the Cristamonadea.

Kofoidia loriculata is a parabasalid symbiont inhabiting the hindgut of the lower termite Paraneotermes simplicicornis. It was initially described as a lophomonad due to its apical tuft of multiple flagella that disintegrate during cell division, but its phylogenetic relationships have not been investigated using molecular evidence. From single cell isolations, we sequenced the small subunit rRNA gene and determined that K. loriculata falls within the Cristamonadea, but is unrelated to other lophomonads. This analysis further demonstrates the polyphyly of the lophomonads and the necessity to re-assess the morphological and cellular evolution of the Cristamonadea.

Correlated SEM, FIB-SEM, TEM, and NanoSIMS imaging of microbes from the hindgut of a lower termite: methods for in situ functional and ecological studies of uncultivable microbes.

The hindguts of lower termites harbor highly diverse, endemic communities of symbiotic protists, bacteria, and archaea essential to the termite's ability to digest wood. Despite over a century of experimental studies, ecological roles of many of these microbes are unknown, partly because almost none can be cultivated. Many of the protists associate with bacterial symbionts, but hypotheses for their respective roles in nutrient exchange are based on genomes of only two such bacteria. To show how the ecological roles of protists and nutrient transfer with symbiotic bacteria can be elucidated by direct imaging, we combined stable isotope labeling (13C-cellulose) of live termites with analysis of fixed hindgut microbes using correlated scanning electron microscopy, focused ion beam-scanning electron microscopy (FIB-SEM), transmission electron microscopy, and high resolution imaging mass spectrometry (NanoSIMS). We developed methods to prepare whole labeled cells on solid substrates, whole labeled cells milled with a FIB-SEM instrument to reveal cell interiors, and ultramicrotome sections of labeled cells for NanoSIMS imaging of 13C enrichment in protists and associated bacteria. Our results show these methods have the potential to provide direct evidence for nutrient flow and suggest the oxymonad protist Oxymonas dimorpha phagocytoses and enzymatically degrades ingested wood fragments, and may transfer carbon derived from this to its surface bacterial symbionts.

Morphology and molecular phylogeny of Staurojoenina mulleri sp. nov. (Trichonymphida, Parabasalia) from the hindgut of the kalotermitid Neotermes jouteli.

Staurojoenina is a large and structurally complex genus of hypermastigont parabasalians found in the hindgut of lower termites. Although several species of Staurojoenina have been described worldwide, all Staurojoenina observed to date in different species of North American termites have been treated as the same species, S. assimilis. Here, we characterize Staurojoenina from the North American termite Neotermes jouteli using light microscopy, scanning electron microscopy, and phylogenetic analysis of small subunit ribosomal RNA, and compare it with S. assimilis from its type host, Incisitermes minor. The basic morphological characteristics of the N. jouteli symbiont, including its abundant bacterial epibionts, are similar as far as they may be compared with existing data from S. assimilis, although not consistently identical. In contrast, we find that they are extremely distantly related at the molecular level, sharing a pairwise similarity of SSU rRNA genes comparable to that seen between different genera or even families of other parabasalians. Based on their evolutionary distance and habitat in different termite genera, we consider the N. jouteli Staurojoenina to be distinct from S. assimilis, and describe a new species, Staurojoenina mulleri, in honor of the pioneering parabasalian researcher, Miklos Muller.

Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts.

Sphingolipids play important roles in plasma membrane structure and cell signaling. However, their lateral distribution in the plasma membrane is poorly understood. Here we quantitatively analyzed the sphingolipid organization on the entire dorsal surface of intact cells by mapping the distribution of (15)N-enriched ions from metabolically labeled (15)N-sphingolipids in the plasma membrane, using high-resolution imaging mass spectrometry. Many types of control experiments (internal, positive, negative, and fixation temperature), along with parallel experiments involving the imaging of fluorescent sphingolipids--both in living cells and during fixation of living cells--exclude potential artifacts. Micrometer-scale sphingolipid patches consisting of numerous (15)N-sphingolipid microdomains with mean diameters of ∼200 nm are always present in the plasma membrane. Depletion of 30% of the cellular cholesterol did not eliminate the sphingolipid domains, but did reduce their abundance and long-range organization in the plasma membrane. In contrast, disruption of the cytoskeleton eliminated the sphingolipid domains. These results indicate that these sphingolipid assemblages are not lipid rafts and are instead a distinctly different type of sphingolipid-enriched plasma membrane domain that depends upon cortical actin.

Fluorinated colloidal gold immunolabels for imaging select proteins in parallel with lipids using high-resolution secondary ion mass spectrometry.

The local abundance of specific lipid species near a membrane protein is hypothesized to influence the protein's activity. The ability to simultaneously image the distributions of specific protein and lipid species in the cell membrane would facilitate testing these hypotheses. Recent advances in imaging the distribution of cell membrane lipids with mass spectrometry have created the desire for membrane protein probes that can be simultaneously imaged with isotope labeled lipids. Such probes would enable conclusive tests to determine whether specific proteins colocalize with particular lipid species. Here, we describe the development of fluorine-functionalized colloidal gold immunolabels that facilitate the detection and imaging of specific proteins in parallel with lipids in the plasma membrane using high-resolution SIMS performed with a NanoSIMS. First, we developed a method to functionalize colloidal gold nanoparticles with a partially fluorinated mixed monolayer that permitted NanoSIMS detection and rendered the functionalized nanoparticles dispersible in aqueous buffer. Then, to allow for selective protein labeling, we attached the fluorinated colloidal gold nanoparticles to the nonbinding portion of antibodies. By combining these functionalized immunolabels with metabolic incorporation of stable isotopes, we demonstrate that influenza hemagglutinin and cellular lipids can be imaged in parallel using NanoSIMS. These labels enable a general approach to simultaneously imaging specific proteins and lipids with high sensitivity and lateral resolution, which may be used to evaluate predictions of protein colocalization with specific lipid species.

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.

Molecular and morphological analysis of the family Calonymphidae with a description of Calonympha chia sp. nov., Snyderella kirbyi sp. nov., Snyderella swezyae sp. nov. and Snyderella yamini sp. nov.

Calonymphids are a group of multinucleate, multiflagellate protists belonging to the order Cristamonadida (Parabasalia) that are found exclusively in the hindgut of termites from the family Kalotermitidae. Despite their impressive morphological complexity and diversity, few species have been formally described and fewer still have been characterized at the molecular level. In this study, four novel species of calonymphids were isolated and characterized: Calonympha chia and Snyderella yamini spp. nov., from Neotermes castaneus and Calcaritermes nearcticus from Florida, USA, and Snyderella kirbyi and Snyderella swezyae, spp. nov., from Calcaritermes nigriceps and Cryptotermes cylindroceps from Colombia. Each of these species was distinguished from its congeners by residing in a distinct host and by differences at the molecular level. Phylogenetic analyses of small subunit (SSU) rDNA indicated that the genera Calonympha and Stephanonympha were probably not monophyletic, though the genus Snyderella, previously only represented by one sequence in molecular analyses, appeared with these new data to be monophyletic. This was in keeping with the traditional evolutionary view of the group in which the morphology of the genus Snyderella is considered to be derived, while that of the genus Stephanonympha is ancestral and therefore probably plesiomorphic.

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.

The inadequacy of morphology for species and genus delineation in microbial eukaryotes: an example from the parabasalian termite symbiont coronympha.

BACKGROUND: For the majority of microbial eukaryotes (protists, algae), there is no clearly superior species concept that is consistently applied. In the absence of a practical biological species concept, most species and genus level delineations have historically been based on morphology, which may lead to an underestimate of the diversity of microbial eukaryotes. Indeed, a growing body of molecular evidence, such as barcoding surveys, is beginning to support the conclusion that significant cryptic species diversity exists. This underestimate of diversity appears to be due to a combination of using morphology as the sole basis for assessing diversity and our inability to culture the vast majority of microbial life. Here we have used molecular markers to assess the species delineations in two related but morphologically distinct genera of uncultivated symbionts found in the hindgut of termites. METHODOLOGY/PRINCIPAL FINDINGS: Using single-cell isolation and environmental PCR, we have used a barcoding approach to characterize the diversity of Coronympha and Metacoronympha symbionts in four species of Incisitermes termites, which were also examined using scanning electron microscopy and light microcopy. Despite the fact that these genera are significantly different in morphological complexity and structural organisation, we find they are two life history stages of the same species. At the same time, we show that the symbionts from different termite hosts show an equal or greater level of sequence diversity than do the hosts, despite the fact that the symbionts are all classified as one species. CONCLUSIONS/SIGNIFICANCE: The morphological information used to describe the diversity of these microbial symbionts is misleading at both the genus and species levels, and led to an underestimate of species level diversity as well as an overestimate of genus level diversity. The genus 'Metacoronympha' is invalid and appears to be a life history stage of Coronympha, while the single recognized species of Coronympha octonaria inhabiting these four termites is better described as four distinct species.

Morphology, phylogeny, and diversity of Trichonympha (Parabasalia: Hypermastigida) of the wood-feeding cockroach Cryptocercus punctulatus.

Trichonympha is one of the most complex and visually striking of the hypermastigote parabasalids-a group of anaerobic flagellates found exclusively in hindguts of lower termites and the wood-feeding cockroach Cryptocercus-but it is one of only two genera common to both groups of insects. We investigated Trichonympha of Cryptocercus using light and electron microscopy (scanning and transmission), as well as molecular phylogeny, to gain a better understanding of its morphology, diversity, and evolution. Microscopy reveals numerous new features, such as previously undetected bacterial surface symbionts, adhesion of post-rostral flagella, and a distinctive frilled operculum. We also sequenced small subunit rRNA gene from manually isolated species, and carried out an environmental polymerase chain reaction (PCR) survey of Trichonympha diversity, all of which strongly supports monophyly of Trichonympha from Cryptocercus to the exclusion of those sampled from termites. Bayesian and distance methods support a relationship between Trichonympha species from termites and Cryptocercus, although likelihood analysis allies the latter with Eucomonymphidae. A monophyletic Trichonympha is of great interest because recent evidence supports a sister relationship between Cryptocercus and termites, suggesting Trichonympha predates the Cryptocercus-termite divergence. The monophyly of symbiotic bacteria of Trichonympha raises the intriguing possibility of three-way co-speciation among bacteria, Trichonympha, and insect hosts.

Surface morphology of Saccinobaculus (Oxymonadida): implications for character evolution and function in oxymonads.

Examination of surface morphology of the oxymonad genus Saccinobaculus from the gut of the wood-feeding cockroach Cryptocercus punctulatus with scanning and transmission electron microscopy reveals several new characters not observable with light microscopy. These include small concavities covering the external surface, a glycocalyx, coated pinocytotic vesicles, and, in one species, unidentified, membrane-bounded organelles with a granular matrix that may represent peroxisomal or mitochondrial derivatives. Unlike representatives of some other oxymonad families, Saccinobaculus lacks extracellular surface structures, a holdfast, and, generally, ectobiotic bacteria. We examined the evolution of these and other characters in light of previously published phylogenies of oxymonads based on molecular data. The presence of concavities in Saccinobaculus and families Pyrsonymphidae and Oxymonadidae strengthens support for a clade comprising these three families. A glycocalyx appears to be a synapomorphy of all oxymonads, and the presence of ectobiotic bacteria also appears to be ancestral to oxymonads, but lost in Saccinobaculus. A holdfast appears to have arisen multiple times. We hypothesize that concavities may play a role in a two-step mechanism for the accumulation and internalization of specific solutes, and that the highly motile and morphologically plastic nature of Saccinobaculus cells limits the possibility of retaining a covering of ectobiotic bacteria.

Morphology and phylogenetic position of Eucomonympha imla (Parabasalia: Hypermastigida).

Eucomonympha imla is a hypermastigote parabasalian found in the gut of the wood-feeding cockroach Cryptocercus punctulatus. It has received little attention since its original description in 1934 as the type species of the genus Eucomonympha and the family Eucomonymphidae. We used light and scanning electron microscopy to characterize surface morphology and organelles, with particular attention to the form of the rostrum, operculum, nucleus, and parabasals. Two previously unrecognized groups of bacterial ectobionts were observed-spirochetes that associate with the flagella and one or more types of rod-shaped bacteria that adhere to the cell surface. The small subunit rRNA (SSU rRNA) sequence was determined from manually isolated cells, and phylogenetic analyses place E. imla in a strongly supported clade with the genera Teranympha and Pseudotrichonympha and three sequences from formally undescribed termite symbionts provisionally assigned to Eucomonympha. Unexpectedly, the Eucomonympha isolates from termites are more closely related to Teranympha than to the type species, suggesting these should not be classified as species of Eucomonympha, despite their morphological similarity to E. imla. Eucomonymphidae fall within a strongly supported Trichonymphida (also including Hoplonymphidae, Trichonymphidae, and Staurojoeninidae), but this clade branches separately from other hypermastigote groups (lophomonads and spirotrichonymphids), suggesting that hypermastigotes are polyphyletic.

Mitochondrial genome of a tertiary endosymbiont retains genes for electron transport proteins.

Mitochondria and plastids originated through endosymbiosis, and subsequently became reduced and integrated with the host in similar ways. Plastids spread between lineages through further secondary or even tertiary endosymbioses, but mitochondria appear to have originated once and have not spread between lineages. Mitochondria are also generally lost in secondary and tertiary endosymbionts, with the single exception of the diatom tertiary endosymbiont of dinoflagellates like Kryptoperidinium foliaceum, where both host and endosymbiont are reported to contain mitochondria. Here we describe the first mitochondrial genes from this system: cytochrome c oxidase 1 (cox1), cytochrome oxidase 3 (cox3), and cytochrome b (cob). Phylogenetic analyses demonstrated that all characterized genes were derived from the pennate diatom endosymbiont, and not the host. We also demonstrated that all three genes are expressed, that cox1 contains spliced group II introns, and that cob and cox3 form an operon, all like their diatom relatives. The endosymbiont mitochondria not only retain a genome, but also express their genes, and are therefore likely involved in electron transport. Ultrastructural examination confirmed the endosymbiont mitochondria retain normal tubular cristae. Overall, these data suggest the endosymbiont mitochondria have not reduced at the genomic or functional level.

Specialized structures in the leaf epidermis of basal angiosperms: morphology, distribution, and homology.

The morphology of specialized structures in the leaf epidermis of 32 species of basal (ANITA: Amborella, Nymphaeales, Illiciales, Trimeniaceae, and Austrobaileyaceae) angiosperms, representing all seven families and 11 of 14 genera, was investigated using light and scanning electron microscopy. Distribution, density, and size of structures were also measured, and character evolution was analyzed. Hydropotes are a synapomorphy of Nymphaeales and ethereal oil cells are a synapomorphy of Austrobaileyales, but uniseriate nonglandular trichomes appear to have arisen independently several times. Specialized structures are frequently characterized by adjacent epidermal cells that have striking similarities in their form and arrangement (i.e., architecture) to subsidiary cells of certain types of stomatal complexes. Additionally, forms intermediate to oil cells and stomata, to trichomes and stomata, and to hydropotes and oil cells are present in some taxa. Thus, all of these specialized structures and their adjacent epidermal cells form complexes that may be homologous with, and evolutionarily derived from stomatal complexes, and the specialized structure, or portion thereof, may be homologous to the stoma or guard mother cell. Improved knowledge of the morphology and evolution of these structures in the earliest branching extant angiosperm lineages has a bearing on many diverse areas of botany.

Stomatal architecture and evolution in basal angiosperms.

Stomatal architecture-the number, form, and arrangement of specialized epidermal cells associated with stomatal guard cells-of 46 species of basal angiosperms representing all ANITA grade families and Chloranthaceae was investigated. Leaf clearings and cuticular preparations were examined with light microscopy, and a sample of 100 stomata from each specimen was coded for stomatal type and five other characters contributing to stomatal architecture. New stomatal types were defined, and many species were examined and illustrated for the first time. Character evolution was examined in light of the ANITA hypothesis using MacClade software. Analysis of character evolution, along with other evidence from this study and evidence from the literature on fossil angiosperms and other seed plant lineages, suggests that the ancestral condition of angiosperms can be described as anomo-stephanocytic, a system in which complexes lacking subdidiaries (anomocytic) intergrade with those having weakly differentiated subsidiaries arranged in a rosette (stephanocytic). From this ancestral condition, tangential divisions of contact cells led to the profusion of different types seen in early fossil angiosperms and Amborellaceae, Austrobaileyales, and derived Chloranthaceae, while the state in Nymphaeales is little modified. Formation of new, derived types by tangential division appears to be a recurrent theme in seed plant evolution.