Genome reduction and horizontal gene transfer in the evolution of Endomicrobia-rise and fall of an intracellular symbiosis with termite gut flagellates.

Bacterial endosymbionts of eukaryotic hosts typically experience massive genome reduction, but the underlying evolutionary processes are often obscured by the lack of free-living relatives. Endomicrobia, a family-level lineage of host-associated bacteria in the phylum Elusimicrobiota that comprises both free-living representatives and endosymbionts of termite gut flagellates, are an excellent model to study evolution of intracellular symbionts. We reconstructed 67 metagenome-assembled genomes (MAGs) of Endomicrobiaceae among more than 1,700 MAGs from the gut microbiota of a wide range of termites. Phylogenomic analysis confirmed a sister position of representatives from termites and ruminants, and allowed to propose eight new genera in the radiation of Endomicrobiaceae. Comparative genome analysis documented progressive genome erosion in the new genus Endomicrobiellum, which comprises all flagellate endosymbionts characterized to date. Massive gene losses were accompanied by the acquisition of new functions by horizontal gene transfer, which led to a shift from a glucose-based energy metabolism to one based on sugar phosphates. The breakdown of glycolysis and many anabolic pathways for amino acids and cofactors in several subgroups was compensated by the independent acquisition of new uptake systems, including an ATP/ADP antiporter, from other gut microbiota. The putative donors are mostly flagellate endosymbionts from other bacterial phyla, including several, hitherto unknown lineages of uncultured Alphaproteobacteria, documenting the importance of horizontal gene transfer in the convergent evolution of these intracellular symbioses. The loss of almost all biosynthetic capacities in some lineages of Endomicrobiellum suggests that their originally mutualistic relationship with flagellates is on its decline.IMPORTANCEUnicellular eukaryotes are frequently colonized by bacterial and archaeal symbionts. A prominent example are the cellulolytic gut flagellates of termites, which harbor diverse but host-specific bacterial symbionts that occur exclusively in termite guts. One of these lineages, the so-called Endomicrobia, comprises both free-living and endosymbiotic representatives, which offers the unique opportunity to study the evolutionary processes underpinning the transition from a free-living to an intracellular lifestyle. Our results revealed a progressive gene loss in energy metabolism and biosynthetic pathways, compensated by the acquisition of new functions via horizontal gene transfer from other gut bacteria, and suggest the eventual breakdown of an initially mutualistic symbiosis. Evidence for convergent evolution of unrelated endosymbionts reflects adaptations to the intracellular environment of termite gut flagellates.

Evaluation of Entomopathogenic Nematode Isolates against Subterranean Termites under Laboratory and Field Conditions.

Background: Termites are a major insect pest affecting agricultural production and woody materials. They cause severe devastation in the ecosystem, and lead to bare soil. This phenomenon causes the soil to become difficult to plow, which in turn leads to a reduction in the productivity of crops. It can cause 100 % yield losses based on crop types, level of the damage, and size of its populations. To manage this pest, different management options have been evaluated in Ethiopia. While insecticide usage is the dominant option, less attention has been given to Entomopathogenic Nematode (EPN) based management options. Therefore, this research was initiated to screen locally collected EPN isolates and evaluate promising isolates under field conditions on maize crop. Methods: 37 EPN isolates were screened under laboratory condition, while two isolates were evaluated at field condition. The screening of EPN isolates was laid out in a completely randomized design, and the field evaluation used a completely randomized block design, and treatments were replicated thrice. Mortality of insect, damaged root, stem, cob, damage severity, foraging termites, and yield of the crop data were collected. Results: The study indicated that all screened EPN isolates caused mortality on termites under laboratory conditions. The isolates achieved complete mortality of the insect pest within 12 days of exposure. The finding indicated that AEH and S#50 were the more pathogenic and virulent isolates on termites under laboratory conditions and taken to field study. The S#50 isolate was most pathogenic and reduced the infestation and severity of the insect pest on the maize crop under field conditions. Conclusions: This result showed that the entomopathogenic nematode isolates have the potential to manage subterranean termites in the maize field. Future studies should be based on collection of local isolates and develop a full package for the virulent isolates.

A Century of Synergy in Termite Symbiosis Research: Linking the Past with New Genomic Insights.

Termites have long been studied for their symbiotic associations with gut microbes. In the late nineteenth century, this relationship was poorly understood and captured the interest of parasitologists such as Joseph Leidy; this research led to that of twentieth-century biologists and entomologists including Cleveland, Hungate, Trager, and Lüscher. Early insights came via microscopy, organismal, and defaunation studies, which led to descriptions of microbes present, descriptions of the roles of symbionts in lignocellulose digestion, and early insights into energy gas utilization by the host termite. Focus then progressed to culture-dependent microbiology and biochemical studies of host-symbiont complementarity, which revealed specific microhabitat requirements for symbionts and noncellulosic mechanisms of symbiosis (e.g., N2 fixation). Today, knowledge on termite symbiosis has accrued exponentially thanks to omic technologies that reveal symbiont identities, functions, and interdependence, as well as intricacies of host-symbiont complementarity. Moving forward, the merging of classical twentieth-century approaches with evolving omic tools should provide even deeper insights into host-symbiont interplay.

First record of gregarine protists (Apicomplexa: Sporozoa) in Asian fungus-growing termite Macrotermes barneyi (Blattaria: Termitidae).

Macrotermes barneyi, widely distributed in southern China, is the major fungus-growing termite in the subfamily Macrotermitinae. It has no flagellated protists in the guts. Here, we report occurrence of gregarine, a protozoan parasite in the digestive tract of M. barneyi. The general morphology and ultrastructure of the gregarine gamonts and syzygies by light micrograph and scanning electron micrograph are presented. SSU rDNA sequence analysis showed that the termite gregarine has the highest identity (90.10%) to that of Gregarina blattarum from cockroaches. Phylogenetic analysis based on the SSU rDNA sequences from diverse insect eugregarines indicated that the gregarine from M. barneyi is phylogenetically close to G. blattarus, L. erratica and G. tropica from Gregarinidae and Leidyanidae families, and may represent a novel species. This study expands our knowledge about the diversity of terrestrial eugregarines parasitizing in termites.

Observation of Giardia sp. in the termite gut of Heterotermes tenuis.

Giardia comprises one genus with several morphologically distinct species described in mammals (including humans, marsupials, rodents), birds, and amphibians. This group of protists provokes diarrhoea diseases in humans and animals worldwide. Transmission of the parasite occurs through the faecal-oral route. Regarding the presence of Giardia in invertebrates, some works have shown that flies can transmit Giardia cysts by contact and transport between contaminated faeces and food. In this way, flies would eventually transmit this parasite. To date, Giardia's presence in the gut of other invertebrates has not been described in the literature. Here we show by first time, using scanning electron microscopy, the presence of Giardia-like trophozoites in the gut of termite Heterotermes tenuis. Two groups of Giardia were found based exclusively on the size and the flange shape of the protozoa: one presented eight flagella, a ventral disc, size, and shape very similar to Giardia intestinalis. In contrast, other cells were smaller and showed some differences in the external morphology. We cannot exclude the possibility that they correspond to the same species and that these differences result from protozoan heterogeneity.

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.

The Complete Protist Symbiont Communities of Coptotermes formosanus and Coptotermes gestroi: Morphological and Molecular Characterization of Five New Species.

Coptotermes formosanus Shiraki and Coptotermes gestroi (Wasmann) (Blattoidea: Rhinotermitidae) are invasive subterranean termite pest species with a major global economic impact. However, the descriptions of the mutualistic protist communities harbored in their respective hindguts remain fragmentary. The C. formosanus hindgut has long been considered to harbor three protist species, Pseudotrichonympha grassii (Trichonymphida), Holomastigotoides hartmanni, and Cononympha (Spirotrichonympha) leidyi (Spirotrichonymphida), but molecular data have suggested that the diversity may be higher. Meanwhile, the C. gestroi community remains undescribed except for Pseudotrichonympha leei. To complete the characterization of these communities, hindguts of workers from both termite species were investigated using single-cell PCR, microscopy, cell counts, and 18S rRNA amplicon sequencing. The two hosts were found to harbor intriguingly parallel protist communities, each consisting of one Pseudotrichonympha species, two Holomastigotoides species, and two Cononympha species. All protist species were unique to their respective hosts, which last shared a common ancestor ~18 MYA. The relative abundances of protist species in each hindgut differed remarkably between cell count data and 18S rRNA profiles, calling for caution in interpreting species abundances from amplicon data. This study will enable future research in C. formosanus and C. gestroi hybrids, which provide a unique opportunity to study protist community inheritance, compatibility, and potential contribution to hybrid vigor.

Nutritional symbiosis and ecology of host-gut microbe systems in the Blattodea.

Cockroaches and termites (Order: Blattodea) have been the subject of substantial research attention for over a century due, in part, to a subset of them having a strong propensity to cohabitate with humans and their structures. Recent research has led to numerous insights into their behavior, physiology, and ecology, as well as their ability to harbor taxonomically diverse microbial communities within their digestive systems, which include taxa that contribute to host growth and development. Further, recent investigations into the physiological and behavioral adaptations that enable recalcitrant polysaccharide digestion and the maintenance of microbial symbionts in cockroaches and termites suggests that symbionts contribute significantly to nutrient provisioning and processing.

Molecular Phylogenetic Position of Hoplonympha natator (Trichonymphea, Parabasalia): Horizontal Symbiont Transfer or Differential Loss?

Hoplonympha natator is an obligate symbiont of Paraneotermes simplicicornis (Kalotermitidae), from southwestern North America. Another Hoplonympha species inhabits Hodotermopsis sjostedti (Archotermopsidae), from montane Southeast Asia. The large phylogenetic and geographical distance between the hosts makes the distribution of Hoplonympha puzzling. Here, we report the phylogenetic position of H. natator from P. simplicicornis through maximum likelihood and Bayesian analysis of 18S rRNA genes. The two Hoplonympha species form a clade with a deep node, making a recent symbiont transfer unlikely. The distribution of Hoplonympha may be due to an ancient transfer or strict vertical inheritance with differential loss from other hosts.

Novel Lineages of Oxymonad Flagellates from the Termite Porotermes adamsoni (Stolotermitidae): the Genera Oxynympha and Termitimonas.

The symbiotic gut flagellates of lower termites form host-specific consortia composed of Parabasalia and Oxymonadida. The analysis of their coevolution with termites is hampered by a lack of information, particularly on the flagellates colonizing the basal host lineages. To date, there are no reports on the presence of oxymonads in termites of the family Stolotermitidae. We discovered three novel, deep-branching lineages of oxymonads in a member of this family, the damp-wood termite Porotermes adamsoni. One tiny species (6-10µm), Termitimonas travisi, morphologically resembles members of the genus Monocercomonoides, but its SSU rRNA genes are highly dissimilar to recently published sequences of Polymastigidae from cockroaches and vertebrates. A second small species (9-13µm), Oxynympha loricata, has a slight phylogenetic affinity to members of the Saccinobaculidae, which are found exclusively in wood-feeding cockroaches of the genus Cryptocercus, the closest relatives of termites, but shows a combination of morphological features that is unprecedented in any oxymonad family. The third, very rare species is larger and possesses a contractile axostyle; it represents a phylogenetic sister group to the Oxymonadidae. These findings significantly advance our understanding of the diversity of oxymonads in termite guts and the evolutionary history of symbiotic digestion.

Molecular Identity of Holomastigotes (Spirotrichonymphea, Parabasalia) with Descriptions of Holomastigotes flavipes n. sp. and Holomastigotes tibialis n. sp.

Holomastigotes is a protist genus (Parabasalia: Spirotrichonymphea) that resides in the hindguts of "lower" termites. It can be distinguished from other parabasalids by spiral flagellar bands that run along the entire length of the cell, an anterior nucleus, a reduced or absent axostyle, the presence of spherical vesicles inside the cells, and the absence of ingested wood particles. Eight species have been described based on their morphology so far, although no molecular data were available prior to this study. We determined the 18S rRNA gene sequences of Holomastigotes from the hindguts of Hodotermopsis sjostedti, Reticulitermes flavipes, Reticulitermes lucifugus, and Reticulitermes tibialis. Phylogenetic analyses placed all sequences in an exclusive and well-supported clade with the type species, Holomastigotes elongatum from R. lucifugus. However, the phylogenetic position of Holomastigotes within the Spirotrichonymphea was not resolved. We describe two new species, Holomastigotes flavipes n. sp. and Holomastigotes tibialis n. sp., inhabiting the hindguts of R. flavipes and R. tibialis, respectively.

Diversity, Roles, and Biotechnological Applications of Symbiotic Microorganisms in the Gut of Termite.

Termites are global pests and can cause serious damage to buildings, crops, and plantation forests. The symbiotic intestinal flora plays an important role in the digestion of cellulose and nitrogen in the life of termites. Termites and their symbiotic microbes in the gut form a synergistic system. These organism work together to digest lignocellulose to make the termites grow on nitrogen deficient food. In this paper, the diversity of symbiotic microorganisms in the gut of termites, including protozoan, spirochetes, actinomycetes, fungus and bacteria, and their role in the digestion of lignocellulose and also the biotechnological applications of these symbiotic microorganisms are discussed. The high efficiency lignocellulose degradation systems of symbiotic microbes in termite gut not only provided a new way of biological energy development, but also has immense prospect in the application of cellulase enzymes. In addition, the study on the symbiotic microorganisms in the gut of termites will also provide a new method for the biological control of termites by the endophytic bacteria in the gut of termites.

Ectosymbiotic bacterial microbiota densely colonize the surface of thelastomatid nematodes in the gut of the wood-feeding cockroach Panesthia angustipennis.

Cockroaches generally harbor thelastomatid nematodes (pinworms) in their gut. In this study, we discovered that the surfaces of two undescribed thelastomatid species in the hindgut of the wood-feeding cockroach Panesthia angustipennis were consistently and densely colonized by bacteria. Epifluorescence microscopy using 4',6-diamidino-2-phenylindole and transmission electron microscopy revealed that several distinct morphotypes of bacteria covered almost the entire body surface of the nematodes in single or multiple layers. Sequencing analysis of 16S rRNA amplicons of either entire nematodes or sections of nematode body surfaces indicated that the associated bacterial microbiota consisted of several dominant phylotypes belonging to either Dysgonomonadaceae (Bacteroidales termite cluster V), Rikennellaceae or Ruminococcaceae. These phylotypes formed clades with sequences previously obtained from cockroach and/or termite guts. Comparisons of the bacterial community structure of the entire cockroach hindgut microbiota vs the nematode-associated microbiota suggested that these dominant bacterial phylotypes preferentially colonized the nematode surface. The two nematode species shared most of the dominant bacterial phylotypes, but the bacterial community structures differed significantly. Colonization by five predominant phylotypes was confirmed by fluorescence in situ hybridization analysis using phylotype-specific probes. Our study provides fundamental information on this previously unknown ectosymbiosis between gut bacteria and thelastomatid pinworms.

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.

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.

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.

The parabasalid symbiont community of Heterotermes aureus: Molecular and morphological characterization of four new species and reestablishment of the genus Cononympha.

The subterranean termite Heterotermes aureus is endemic to arid regions of southwestern USA and northern Mexico. Like other termites in the family Rhinotermitidae, it harbors a community of protists (Phylum Parabasalia) in its hindgut that aid in cellulose digestion. We investigated the hindgut community of H. aureus using light microscopy, single cell isolation, and high throughput amplicon sequencing. Here we describe four new parabasalid species from the classes Trichonymphea and Spirotrichonymphea. Three of the new species include Pseudotrichonympha aurea (Trichonymphea), Holomastigotoides aureus, and Holomastigotoides oxyrhynchus (Spirotrichonymphea). The fourth new species is a Spirotrichonympha-like protist for which we reinstate the genus Cononympha and describe under the name Cononympha aurea (Spirotrichonymphea). We also used high throughput amplicon sequencing with custom primers on DNA from fresh and ethanol preserved termites collected across the southwest USA and Mexico to investigate population-level differences in hindgut community composition. We report that the community is highly similar across populations: no additional parabasalid species were identified in any of the H. aureus specimens, but several specimens appeared to lack either C. aurea or H. oxyrhynchus.

Discovery of ectosymbiotic Endomicrobium lineages associated with protists in the gut of stolotermitid termites.

The genus Endomicrobium is a dominant bacterial group in the gut of lower termites, and most phylotypes are intracellular symbionts of gut protists. Here we report the discovery of Endomicrobium ectosymbionts of termite gut protists. We found that bristle-like Endomicrobium cells attached to the surface of spirotrichosomid protist cells inhabiting the termite Stolotermes victoriensis. Transmission electron microscopy revealed that a putative Endomicrobium cell likely attached to the protist surface via a protrusion from the tip of the bacterium. A phylotype, sharing 98.9% 16S rRNA sequence identity with the Endomicrobium ectosymbionts of the spirotrichosomid protists, was also found on the cell surface of the protist Trichonympha magna in the gut of the termite Porotermes adamsoni. We propose the novel species 'Candidatus Endomicrobium superficiale' for these bacteria. T. magna simultaneously harboured another Endomicrobium ectosymbiont that shared 93.5-94.2% 16S rRNA sequence identities with 'Ca. Endomicrobium superficiale'. Furthermore, Spirotrichonympha-like protists in P. adamsoni guts were associated with an Endomicrobium phylotype that possibly attached to the host flagella. A phylogenetic analysis suggested that these ectosymbiotic lineages have evolved multiple times from free-living Endomicrobium lineages and are relatively distant from the endosymbionts. Our results provide novel insights into the ecology and evolution of the Endomicrobium.

RNA interference unveils the importance of Pseudotrichonympha grassii cellobiohydrolase, a protozoan exoglucanase, in termite cellulose degradation.

Based on prior work, a cellulase from glycosyl hydrolase family 7 (GHF7) was identified and found to be expressed at a high level in Coptotermes formosanus. To determine the function of GHF7 family members in vivo, we used RNA interference (RNAi) to functionally analyse the exoglucanase gene Pseudotrichonympha grassii cellobiohydrolase gene (PgCBH), which was highly expressed in Pseudotrichonympha grassii, a flagellate found in the hindgut of C. formosanus. In this study, the expression level of PgCBH was down-regulated by RNAi, causing the death of P. grassii, but no effect was observed for other flagellates found in C. formosanus. RNAi also resulted in significantly reduced exoglucanase activity, and no effect was observed for endoglucanase and ß-glucosidase activities. This result demonstrated that the PgCBH gene plays a role in the protist lignocellulolytic process and is also important for host survival. PgCBH can be used as a target gene and has potential as a bioinsecticide for use against termites.

Comparison of Intracellular "Ca. Endomicrobium Trichonymphae" Genomovars Illuminates the Requirement and Decay of Defense Systems against Foreign DNA.

"Candidatus Endomicrobium trichonymphae" (Bacteria; Elusimicrobia) is an obligate intracellular symbiont of the cellulolytic protist genus Trichonympha in the termite gut. A previous genome analysis of "Ca Endomicrobium trichonymphae" phylotype Rs-D17 (genomovar Ri2008), obtained from a Trichonympha agilis cell in the gut of the termite Reticulitermes speratus, revealed that its genome is small (1.1 Mb) and contains many pseudogenes; it is in the course of reductive genome evolution. Here we report the complete genome sequence of another Rs-D17 genomovar, Ti2015, obtained from a different T. agilis cell present in an R. speratus gut. These two genomovars share most intact protein-coding genes and pseudogenes, showing 98.6% chromosome sequence similarity. However, characteristic differences were found in their defense systems, which comprised restriction-modification and CRISPR/Cas systems. The repertoire of intact restriction-modification systems differed between the genomovars, and two of the three CRISPR/Cas loci in genomovar Ri2008 are pseudogenized or missing in genomovar Ti2015. These results suggest relaxed selection pressure for maintaining these defense systems. Nevertheless, the remaining CRISPR/Cas system in each genomovar appears to be active; none of the "spacer" sequences (112 in Ri2008 and 128 in Ti2015) were shared whereas the "repeat" sequences were identical. Furthermore, we obtained draft genomes of three additional endosymbiotic Endomicrobium phylotypes from different host protist species, and discovered multiple, intact CRISPR/Cas systems in each genome. Collectively, unlike bacteriome endosymbionts in insects, the Endomicrobium endosymbionts of termite-gut protists appear to require defense against foreign DNA, although the required level of defense has likely been reduced during their intracellular lives.