Extra-pair paternity in the wood-feeding cockroach Cryptocercus punctulatus Scudder: Social but not genetic monogamy.

Subsocial Cryptocercus cockroaches are the sister group to termites and considered to be socially monogamous. Because genetic monogamy is a suggested requirement for evolution of cooperative breeding/eusociality, particularly in hymenopterans, clarification of the mating biology of Cryptocercus would help illuminate evolutionary trends in eusocial insects. To investigate possible extra-pair paternity in C. punctulatus, microsatellite markers were used to analyse offspring parentage, the stored sperm in females and results of experimental manipulation of sperm competition. Extra-pair paternity was common in field-collected families, but a lack of maternal alleles in several nymphs suggests sampling error or adoption. Isolating prereproductive pairs and assaying subsequently produced nymphs confirmed that nymphs lacked alleles from the pair male in 40% of families, with extra-pair male(s) siring 27%-77% of nymphs. Sperm of extra-pair males was detected in the spermatheca of 51% of paired prereproductive females. Mate switching and surgical manipulation of male mating ability indicated a tendency towards last male sperm precedence. Overall, the results demonstrate that about half of young females are serially monogamous during their maturational year, but bond, overwinter and produce their only set of offspring in company of the last mated male (=pair male). Repeated mating by the pair male increases the number of nymphs sired, but because many females use stored sperm of previous copulatory partners to fertilize eggs, pair males extend parental care to unrelated nymphs. The results suggest that genetic monogamy either developed in the termite ancestor after splitting from the Cryptocercus lineage, or that genetic monogamy may not be a strict prerequisite for the evolution of termite eusociality.

Distribution of Karyotypes of the Cryptocercus punctulatus Species Complex (Blattodea: Cryptocercidae) in Great Smoky Mountains National Park.

During the period between 1999 and 2006, wood-feeding cockroaches in the Cryptocercus punctulatus Scudder species complex were collected throughout Great Smoky Mountains National Park, USA. The chromosome numbers of insects from 59 sites were determined, and phylogenetic analyses were performed based on mitochondrial COII and nuclear ITS2 DNA. The distribution of the three male karyotypes found in the park (2n = 37, 39, and 45) is mapped and discussed in relation to recent disturbances and glacial history. Clades of the three karyotype groups meet near the ridgeline separating North Carolina from Tennessee in the center of the park, suggesting that these may have originated from separate lower elevation refugia after the last glacial maximum. The timing of divergence and a significant correlation between elevation difference and genetic distance in two of the clades supports this hypothesis. The ecological role of the cockroaches in the park is discussed.

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.

The role of host phylogeny varies in shaping microbial diversity in the hindguts of lower termites.

The hindguts of lower termites and Cryptocercus cockroaches are home to a distinct community of archaea, bacteria, and protists (primarily parabasalids and some oxymonads). Within a host species, the composition of these hindgut communities appears relatively stable, but the evolutionary and ecological factors structuring community composition and stability are poorly understood, as are differential impacts of these factors on protists, bacteria, and archaea. We analyzed the microbial composition of parabasalids and bacteria in the hindguts of Cryptocercus punctulatus and 23 species spanning 4 families of lower termites by pyrosequencing variable regions of the small-subunit rRNA gene. Especially for the parabasalids, these data revealed undiscovered taxa and provided a phylogenetic basis for a more accurate understanding of diversity, diversification, and community composition. The composition of the parabasalid communities was found to be strongly structured by the phylogeny of their hosts, indicating the importance of historical effects, although exceptions were also identified. Particularly, spirotrichonymphids and trichonymphids likely were transferred between host lineages. In contrast, host phylogeny was not sufficient to explain the majority of bacterial community composition, but the compositions of the Bacteroidetes, Elusimicrobia, Tenericutes, Spirochaetes, and Synergistes were structured by host phylogeny perhaps due to their symbiotic associations with protists. All together, historical effects probably resulting from vertical inheritance have had a prominent role in structuring the hindgut communities, especially of the parabasalids, but dispersal and environmental acquisition have played a larger role in community composition than previously expected.

Altricial development in subsocial cockroach ancestors: foundation for the evolution of phenotypic plasticity in termites.

Basal termites possess two developmental features that eusocial Hymenoptera lack: the majority of colony members are juveniles whose somatic and reproductive development is temporarily or permanently suspended, and individual development is characterized by extreme phenotypic plasticity. An examination of the literature indicates that the basis for these unique ontogenetic characters is not the prolongation of a pronymphal stage into postembryonic development, as recently suggested. Like other hemimetabolous insects, termites have three embryonic cuticles, and the pronymphal (EC3) cuticle is shed during or shortly after hatch. Nonetheless, a different developmental landmark, dorsal closure, occurs later during embryogenesis in termites than it does in their cockroach relatives, clearly indicating ontogenetic repatterning from an ancestral state. An alternate hypothesis for the origin of isopteran phenotypic plasticity becomes apparent if we remain focused on the phylogenetic and social context of termite evolution. Altricial development occurs in both vertebrate and invertebrate taxa, evolves in response to the parental environment, and is displayed by two distantly related, biparental, wood-feeding cockroaches, including Cryptocercus, the sister-group to termites. It is therefore likely the condition was present in subsocial termite ancestors, and played a complex, multidimensional role in the transition to eusociality. Most relevant to current arguments is that a shift in responsibility for the care of altricial dependents, from parents to the first nutritionally independent nymphs in the family (alloparents), resulted in the developmental stasis of alloparents at a relatively young age. Because early instar cockroaches are not metamorphically competent, these young alloparents would have provided a novel developmental template on which selection could act.

Inheritance and diversification of symbiotic trichonymphid flagellates from a common ancestor of termites and the cockroach Cryptocercus.

Cryptocercus cockroaches and lower termites harbour obligate, diverse and unique symbiotic cellulolytic flagellates in their hindgut that are considered critical in the development of social behaviour in their hosts. However, there has been controversy concerning the origin of these symbiotic flagellates. Here, molecular sequences encoding small subunit rRNA and glyceraldehyde-3-phosphate dehydrogenase were identified in the symbiotic flagellates of the order Trichonymphida (phylum Parabasalia) in the gut of Cryptocercus punctulatus and compared phylogenetically to the corresponding species in termites. In each of the monophyletic lineages that represent family-level groups in Trichonymphida, the symbionts of Cryptocercus were robustly sister to those of termites. Together with the recent evidence for the sister-group relationship of the host insects, this first comprehensive study comparing symbiont molecular phylogeny strongly suggests that a set of symbiotic flagellates representative of extant diversity was already established in an ancestor common to Cryptocercus and termites, was vertically transmitted to their offspring, and subsequently became diversified to distinct levels, depending on both the host and the symbiont lineages.

Altricial development in subsocial wood-feeding cockroaches.

Species in the wood-feeding genus Salganea within the cockroach subfamily Panesthiinae (Blaberidae) typically live in biparental families; their first instars suffer high mortality when removed from adults, and in at least one species, adults are known to feed neonates on oral liquids. In the closely related gregarious wood-feeding genus Panesthia , no parental interaction with offspring is known. We compared the external morphology of first instars of these two genera and found that eye development and cuticular pigmentation at hatching are correlated with social structure. First instars of Panesthia have a dark cuticle and well-developed eyes. First instars of Salganea have a pale, transparent cuticle, and eyes significantly smaller than those of Panesthia relative to adult eye size. The body size of the first-instar of Salganea esakii is significantly smaller than that of Panesthia angustipennis spadica , relative to adult body size (24.0 and 27.4% of adult head-capsule width, respectively), but a more general survey suggests that, overall, neonate size may be similar in the two genera. We compared the first instars of these two taxa of Panesthiinae to those in the biparental, wood-feeding cockroach Cryptocercus (Cryptocercidae) and discuss how decreased investment in both integumentary and ocular development in subsocial cockroaches parallels that seen in altricial vertebrates.

Wasp Size and Prey Load in Cerceris fumipennis (Hymenoptera, Crabronidae): Implications for Biosurveillance of Pest Buprestidae.

The relationship between predator and prey size was studied in the buprestid hunting wasp Cerceris fumipennis Say in eight widely distributed nesting aggregations in North Carolina, USA. Initial work indicated a significant linear relationship between wasp head width and wasp wet weight; thus, head width was used to estimate wasp body mass in subsequent studies. Prey loads of hunting females was studied by measuring the head width of the wasp, then identifying and weighing the prey item brought back to the nest. There was significant variation in wasp size among nesting aggregations; the average estimated wasp body mass in one site was double that in another. Prey weight varied with wasp weight, but larger wasps had a slight tendency to carry proportionally larger prey. Beetles captured by large wasps (≥120 mg) were significantly more variable in weight than those taken by small wasps (<80 mg). All but the smallest wasps could carry more than their own body weight. Prey loads ranged from 4.8⁻150.2% of wasp weight. Evidence suggests that small wasps bring back more of the economically important buprestid genus Agrilus and thus would be most efficient in biosurveillance for pest buprestids.

A Crucial Caste Regulation Gene Detected by Comparing Termites and Sister Group Cockroaches.

Sterile castes are a defining criterion of eusociality; investigating their evolutionary origins can critically advance theory. In termites, the soldier caste is regarded as the first acquired permanently sterile caste. Previous studies showed that juvenile hormone (JH) is the primary factor inducing soldier differentiation, and treatment of workers with artificial JH can generate presoldier differentiation. It follows that a shift from a typical hemimetabolous JH response might be required for soldier formation during the course of termite evolution within the cockroach clade. To address this possibility, analysis of the role of JH and its signaling pathway was performed in the termite Zootermopsis nevadensis and compared with the wood roach Cryptocercus punctulatus, a member of the sister group of termites. Treatment with a JH analog (JHA) induced a nymphal molt in C. punctulatus RNA interference (RNAi) of JH receptor Methoprene tolerant (Met) was then performed, and it inhibited the presoldier molt in Z. nevadensis and the nymphal molt in C. punctulatus Knockdown of Met in both species inhibited expression of 20-hydroxyecdysone (20E; the active form of ecdysone) synthesis genes. However, in Z. nevadensis, several 20E signaling genes were specifically inhibited by Met RNAi. Consequently, RNAi of these genes were performed in JHA-treated termite individuals. Knockdown of 20E signaling and nuclear receptor gene, Hormone receptor 39 (HR39/FTZ-F1ß) resulted in newly molted individuals with normal worker phenotypes. This is the first report of the JH-Met signaling feature in termites and Cryptocercus JH-dependent molting activation is shared by both taxa and mediation between JH receptor and 20E signalings for soldier morphogenesis is specific to termites.

Parallel and Gradual Genome Erosion in the Blattabacterium Endosymbionts of Mastotermes darwiniensis and Cryptocercus Wood Roaches.

Almost all examined cockroaches harbor an obligate intracellular endosymbiont, Blattabacterium cuenoti. On the basis of genome content, Blattabacterium has been inferred to recycle nitrogen wastes and provide amino acids and cofactors for its hosts. Most Blattabacterium strains sequenced to date harbor a genome of ∼630 kbp, with the exception of the termite Mastotermes darwiniensis (∼590 kbp) and Cryptocercus punctulatus (∼614 kbp), a representative of the sister group of termites. Such genome reduction may have led to the ultimate loss of Blattabacterium in all termites other than Mastotermes. In this study, we sequenced 11 new Blattabacterium genomes from three species of Cryptocercus in order to shed light on the genomic evolution of Blattabacterium in termites and Cryptocercus. All genomes of Cryptocercus-derived Blattabacterium genomes were reduced (∼614 kbp), except for that associated with Cryptocercus kyebangensis, which comprised 637 kbp. Phylogenetic analysis of these genomes and their content indicates that Blattabacterium experienced parallel genome reduction in Mastotermes and Cryptocercus, possibly due to similar selective forces. We found evidence of ongoing genome reduction in Blattabacterium from three lineages of the C. punctulatus species complex, which independently lost one cysteine biosynthetic gene. We also sequenced the genome of the Blattabacterium associated with Salganea taiwanensis, a subsocial xylophagous cockroach that does not vertically transmit gut symbionts via proctodeal trophallaxis. This genome was 632 kbp, typical of that of nonsubsocial cockroaches. Overall, our results show that genome reduction occurred on multiple occasions in Blattabacterium, and is still ongoing, possibly because of new associations with gut symbionts in some lineages.

Harmonia axyridis (Coleoptera: Coccinellidae) in buildings: relationship between body height and crevice size allowing entry.

Although the introduced lady beetle Harmonia axyridis (Pallas) (Coleoptera: Coc-cinellidae) is an important predator of aphids in a variety of crop systems during the growing season, it is often a pest in fall and winter when it enters buildings seeking overwintering sites. One of the primary recommendations for managing this annual influx is to prevent beetle entry by caulking or otherwise filling potential entry points in buildings. The goal of this study was to determine how small a gap the beetles are able to enter in choice and no-choice studies by experimentally exploiting their behavioral tendency to seek dark shelters at cool temperatures. Within the size range of adults collected in central North Carolina in 2003 (1.99-3.29 mm body height), no beetles entered a 2-mm access during no-choice experiments. Most (83%) entered a 3-mm gap; those failing to cross the 3-mm threshold were significantly larger than those that traversed it. In choice experiments, 98.2% of beetles entered shelters. As in the previous study, no beetles entered shelters with 2-mm gaps. Significantly fewer were found in shelters with 3-mm entrances than in those with 4- or 5-mm access; beetles that entered 3-mm gaps were significantly smaller than the remainder of the test population. Although no H. axyridis crossed a 2-mm threshold in either experiment, a gap of this size may nonetheless allow admission if it has flexible borders (e.g., foam weather stripping); beetles were observed attempting forced entry into too-small crevices.

What Kills the Hindgut Flagellates of Lower Termites during the Host Molting Cycle?

Subsocial wood feeding cockroaches in the genus Cryptocercus, the sister group of termites, retain their symbiotic gut flagellates during the host molting cycle, but in lower termites, closely related flagellates die prior to host ecdysis. Although the prevalent view is that termite flagellates die because of conditions of starvation and desiccation in the gut during the host molting cycle, the work of L.R. Cleveland in the 1930s through the 1960s provides a strong alternate hypothesis: it was the changed hormonal environment associated with the origin of eusociality and its concomitant shift in termite developmental ontogeny that instigates the death of the flagellates in termites. Although the research on termite gut microbial communities has exploded since the advent of modern molecular techniques, the role of the host hormonal environment on the life cycle of its gut flagellates has been neglected. Here Cleveland's studies are revisited to provide a basis for re-examination of the problem, and the results framed in the context of two alternate hypotheses: the flagellate symbionts are victims of the change in host social status, or the flagellates have become incorporated into the life cycle of the eusocial termite colony. Recent work on parasitic protists suggests clear paths for exploring these hypotheses and for resolving long standing issues regarding sexual-encystment cycles in flagellates of the Cryptocercus-termite lineage using molecular methodologies, bringing the problem into the modern era.

Molecular phylogeny of Cryptocercus wood-roaches based on mitochondrial COII and 16S sequences, and chromosome numbers in Palearctic representatives.

Woodroaches of the genus Cryptocercus are subsocial and xylophagous cockroaches, distributed in North America and Asia. Studies on male chromosome number in Nearctic species have shown that diploid numbers vary from 2n=37 to 2n=47; numbers from Palearctic species were heretofore unknown. Two hypotheses have been proposed to explain the varying number of chromosomes among Nearctic species: the serial reduction hypothesis, and the parallel scenario. We performed phylogenetic analyses of the COII gene in these species and found evidence for the topology (47(45(43(39,37), which is congruent with the serial reduction hypothesis. We also determined chromosome numbers for the first time in Palearctic species, and found Cryptocercus primarius and Cryptocercus relictus to have relatively low chromosome numbers (2n=17-21) compared to their Nearctic relatives. Finally, our study determined the phylogenetic position of Cryptocercus primarius among other Asian taxa.

Biogeography and Phylogeny of Wood-feeding Cockroaches in the Genus Cryptocercus.

Subsocial, xylophagous cockroaches of the genus Cryptocercus exhibit a disjunct distribution, with representatives in mature montane forests of North America, China, Korea and the Russian Far East. All described species are wingless and dependent on rotting wood for food and shelter at all stages of their life cycle; consequently, their distribution is tied to that of forests and strongly influenced by palaeogeographical events. Asian and American lineages form distinct monophyletic groups, comprised of populations with complex geographic substructuring. We review the phylogeny and distribution of Cryptocercus, and discuss splitting events inferred from molecular data.

Antennal cropping during colony foundation in termites.

The literature on pairing and mating behavior in termites indicates that a number of distal antennal segments in dealates of both sexes are often removed during colony foundation, with terms such as amputation, mutilation and cannibalism typically employed to report the phenomenon. Here we propose the use of the phrase 'antennal cropping' to describe the behavior, and assess naturally occurring levels of its occurrence by comparing the number of antennal segments in museum specimens of alates and dealates in 16 species of Australian termites (four families), supplemented by analyzing published data on Coptotermes gestroi. Dealates had significantly fewer antennal segments than alates in 14 of the 16 termite species, with both exceptions belonging to the family Termitidae. Levels of antennal cropping were not significantly different between the sexes but did vary by family. Dealates in the Kalotermitidae removed the most segments (41.3%) and those in the Termitidae removed the fewest (8.9%). We discuss the biological significance of this phylogenetically widespread termite behavior, and suggest that controlled antennal cropping is not only a normal part of their behavioral repertoire but also a key influence that changes the conduct and physiology of the royal pair during the initial stages of colony foundation.

Phylogenetic analysis of cellulolytic enzyme genes from representative lineages of termites and a related cockroach.

The relationship between xylophagous termites and the protists resident in their hindguts is a textbook example of symbiosis. The essential steps of lignocellulose degradation handled by these protists allow the host termites to thrive on a wood diet. There has never been a comprehensive analysis of lignocellulose degradation by protists, however, as it has proven difficult to establish these symbionts in pure culture. The trends in lignocellulose degradation during the evolution of the host lineage are also largely unknown. To clarify these points without any cultivation technique, we performed meta-expressed sequence tag (EST) analysis of cDNA libraries originating from symbiotic protistan communities in four termite species and a wood-feeding cockroach. Our results reveal the establishment of a degradation system with multiple enzymes at the ancestral stage of termite-protistan symbiosis, especially GHF5 and 7. According to our phylogenetic analyses, the enzymes comprising the protistan lignocellulose degradation system are coded not only by genes innate to the protists, but also genes acquired by the protists via lateral transfer from bacteria. This gives us a fresh perspective from which to understand the evolutionary dynamics of symbiosis.

The fine structure of colleterial glands in two cockroaches and three termites, including a detailed study of Cryptocercus punctulatus (Blattaria, Cryptocercidae) and Mastotermes darwiniensis (Isoptera, Mastotermitidae).

The colleterial glands of insects are organs associated with the female genital apparatus. In cockroaches, these glands produce secretions that cover two parallel rows of eggs during oviposition, and in oviparous species, these secretions become the tanned, sculpted, rigid outer casing of the ootheca. The goal of this study was to compare the gross anatomy of the colleterial glands and the ultrastructure of their component tubules in the phylogenetically significant genera Cryptocercus (Blattaria) and Mastotermes (Isoptera). Recent studies indicate that cockroaches in the genus Cryptocercus are the sister group of termites, and Mastotermes is the only termite known to produce a cockroach-like ootheca. One additional oviparous cockroach, Therea, and two additional termites, Zootermopsis and Pseudacanthotermes, were also examined. As in other cockroaches, the colleterial glands of Cryptocercus and Therea are asymmetrical, with a well developed bipartite left gland and a smaller right gland. In the termites Mastotermes, Zootermopsis, and Pseudacanthotermes, the colleterial glands are composed of a well-developed, paired, anterior gland and a small posterior gland; histological staining and cytological evidence suggest that these are homologues of the left and the right colleterial glands of cockroaches, respectively. At the ultrastructural level, colleterial gland tubules are made of cells belonging to a modified class 1 type cell in the cockroaches, in Mastotermes, and in Zootermopsis; the latter lays its eggs singly, without a surrounding ootheca-like structure. In the advanced termite Pseudacanthotermes, the tubules are made of secretory units belonging to the class 3 cell type. This study demonstrates that the cytological characteristics of colleterial glands in basal termites are similar to those of cockroaches, whether the termite secretes an oothecal casing that covers two parallel rows of eggs, as in Mastotermes, or lays its eggs singly, as in Zootermopsis. The function of colleterial glands in non-mastotermitid termites is unknown.

Infection of Harmonia axyridis (Coleoptera: Coccinellidae) by Hesperomyces virescens (Ascomycetes: Laboulbeniales): role of mating status and aggregation behavior.

The ectoparasitic fungus Hesperomyces virescens was studied on Harmonia axyridis in North Carolina, in the southeastern United States. A primary goal was to investigate transmission of the disease by examining the correlation between the pattern of fungal infection and seasonal change in host behavior. Beetles were collected as they arrived at their winter quarters at two sites; in one site they were also subsampled at mid- and late winter. Insects were sexed and weighed, fungal thalli were counted, and their location on the host body mapped; spermathecae of females were examined for sperm. Infection levels varied between sites, differed significantly between the sexes in one site but not the other, and increased by approximately 40% during winter. The distribution of thalli on the body changed seasonally, in concert with behavioral changes in the host. At fall flight, thalli were found most often on the posterior elytra of mated females, virgin females, and males. This is suggestive that the disease had been spread among both sexes via successful and failed copulation attempts; however, the relatively low incidence of infection on the male venter does not fit the sexual transmission scenario. During winter, thallus location shifts in concert with beetle aggregation behavior, with infections more often located on the head and legs. Fresh weight of beetles decreased by approx. 20% during winter, but was not affected by disease status. Prior to spring flight, uninfected females were preferred as mating partners, but the probable relationship between female age and infection status complicates interpretation of the data.

Identification and characterization of ectosymbionts of distinct lineages in Bacteroidales attached to flagellated protists in the gut of termites and a wood-feeding cockroach.

Bacterial attachments to nearly the entire surface of flagellated protists in the guts of termites and the wood-feeding cockroach Cryptocercus are often observed. Based on the polymerase chain reaction-amplified 16S rRNA gene sequences, we investigated the phylogenetic relationships of the rod-shaped, attached bacteria (ectosymbionts) of several protist species from five host taxa and confirmed their identity by fluorescence in situ hybridizations. These ectosymbionts are affiliated with the order Bacteroidales but formed three distinct lineages, each of which may represent novel bacterial genera. One lineage consisted of the closely related ectosymbionts of two species of the protist genus Devescovina (Cristamonadida). The second lineage comprised three phylotypes identified from the protist Streblomastix sp. (Oxymonadida). The third lineage included ectosymbionts of the three protist genera Hoplonympha, Barbulanympha and Urinympha in the family Hoplonymphidae (Trichonymphida). The ultrastructural observations indicated that these rod-shaped ectosymbionts share morphological similarities of their cell walls and their point of attachment with the protist but differ in shape. Elongated forms of the ectosymbionts appeared in all the three lineages. The protist cells Streblomastix sp. and Hoplonympha sp. display deep furrows and vane-like structures, but these impressive structures are probably evolutionarily convergent because both the host protists and their ectosymbionts are distantly related.