Bacteriome-associated endosymbionts of the green rice leafhopper <Emphasis Type="Italic">Nephotettix cincticeps</Emphasis> (Hemiptera: Cicadellidae)

The green rice leafhopper Nephotettix cincticeps (Uhler) is a commonly distributed pest of rice in East Asia. Early histological studies describe the presence of two bacteriome-associated symbionts and a rickettsial microorganism in N. cincticeps, but their microbiological affiliations have been elusive. We identified these bacterial symbionts using modern microbiological techniques. Cloning and sequencing of the 16S ribosomal RNA gene from dissected bacteriomes yielded two major and a minor bacterial sequences: a major sequence was placed in the Bacteroidetes clade of Sulcia muelleri, an ancient symbiont lineage associated with diverse hemipteran insects; another major sequence was allied to a β-proteobacterial sequence from a leafhopper Matsumuratettix hiroglyphicus; the minor sequence fell in the α-proteobacterial genus Rickettsia. In situ hybridization and transmission electron microscopy showed that the Sulcia symbiont and the β-proteobacterial symbiont are harbored within different types of bacteriocytes that constitute the outer and inner regions of the bacteriome, respectively. Oral administration of tetracycline to nymphal N. cincticeps resulted in retarded growth, high mortality rates, and failure in adult emergence, suggesting important biological roles of the symbionts for the host insect. The designation Candidatus Nasuia deltocephalinicola is proposed for the β-proteobacterial symbiont clade associated with N. cincticeps and allied leafhoppers of the subfamily Deltocephalinae.     

Coexistence of multiple proteobacterial endosymbionts in the gills of the wood-boring Bivalve Lyrodus pedicellatus (Bivalvia: Teredinidae)

Wood-boring bivalves of the family Teredinidae (commonly called shipworms) are known to harbor dense populations of gram-negative bacteria within specialized cells (bacteriocytes) in their gills. These symbionts are thought to provide enzymes, e.g., cellulase and dinitrogenase, which assist the host in utilizing wood as a primary food source. A cellulolytic, dinitrogen-fixing bacterium, Teredinibacter turnerae, has been isolated from the gill tissues of numerous teredinid bivalves and has been proposed to constitute the sole or predominant symbiont of this bivalve family. Here we demonstrate that one teredinid species, Lyrodus pedicellatus, contains at least four distinct bacterial 16S rRNA types within its gill bacteriocytes, one of which is identical to that of T. turnerae. Phylogenetic analyses indicate that the three newly detected ribotypes are derived from gamma proteobacteria that are related to but distinct (>6.5% sequence divergence) from T. turnerae. In situ hybridizations with 16S rRNA-directed probes demonstrated that the pattern of occurrence of symbiont ribotypes within bacteriocytes was predictable and specific, with some bacteriocytes containing two symbiont ribotypes. However, only two of the six possible pairwise combinations of the four ribotypes were observed to cooccur within the same host cells. The results presented here are consistent with the existence of a complex multiple symbiosis in this shipworm species.     

Characterization and evolution of two bacteriome‐inhabiting symbionts in cixiid planthoppers (Hemiptera: Fulgoromorpha: Pentastirini)

Like other plant sap‐sucking insects, planthoppers within the family Cixiidae (Insecta: Hemiptera: Fulgoromorpha) host a diversified microbiota. We report the identification and first molecular characterization of symbiotic bacteria in cixiid planthoppers (tribe: Pentastirini). Using universal eubacterial primers we first screened the eubacterial 16S rRNA sequences in Pentastiridius leporinus (Linnaeus) with PCR amplification, cloning, and restriction fragment analysis. We identified three main 16S rRNA sequences that corresponded to a Wolbachia bacterium, a plant pathogenic bacterium, and a novel gammaproteobacterial symbiont. A fourth bacterial species affiliated with ‘Candidatus Sulcia muelleri’ was detected in PCR assays using primers specific for the Bacteroidetes. Within females of two selected cixiid planthoppers, P. leporinus and Oliarus filicicola, fluorescence In situ hybridization analysis and transmission electron microscopy observations showed that ‘Ca. Sulcia muelleri’ and the novel gammaproteobacterial symbiont were housed in separate bacteriomes. Phylogenetic analysis revealed that both of these symbionts occurred in at least four insect genera within the tribe Pentastirini. ‘Candidatus Purcelliella pentastirinorum’ was proposed as the novel gammaproteobacterial symbiont.     

Biotype-dependent secondary symbiont communities in sympatric populations of Bemisia tabaci

The sweet potato whitefly, Bemisia tabaci, harbors Portiera aleyrodidarum, an obligatory symbiotic bacterium, as well as several secondary symbionts including Rickettsia, Hamiltonella, Wolbachia, Arsenophonus, Cardinium and Fritschea, the function of which is unknown. Bemisia tabaci is a species complex composed of numerous biotypes, which may differ from each other both genetically and biologically. Only the B and Q biotypes have been reported from Israel. Secondary symbiont infection frequencies of Israeli laboratory and field populations of B. tabaci from various host plants were determined by PCR, in order to test for correlation between bacterial composition to biotype and host plant. Hamiltonella was detected only in populations of the B biotype, while Wolbachia and Arsenophonus were found only in the Q biotype (33% and 87% infection, respectively). Rickettsia was abundant in both biotypes. Cardinium and Fritschea were not found in any of the populations. No differences in secondary symbionts were found among host plants within the B biotype; but within the Q biotype, all whiteflies collected from sage harboured both Rickettsia and Arsenophonus, an infection frequency which was significantly higher than those found in association with all other host plants. The association found between whitefly biotypes and secondary symbionts suggests a possible contribution of these bacteria to host characteristics such as insecticide resistance, host range, virus transmission and speciation.     

Buchnera aphidicola of the birch blister aphid,Hamamelistes betulinus (Horváth, 1896) (Insecta,Hemiptera, Aphididae: Hormaphidinae): molecular characterization,transmission between generations and its geographic significance

The birch blister aphid Hamamelistes betulinus, like most aphids, is host to obligate symbiotic bacterium Buchnera aphidicola. Ultrastructural and molecular analyses did not reveal the presence of secondary symbionts in the body of H. betulinus. The bacteria Buchnera aphidicola are transmitted to the next generation vertically (maternally). The bacteria released from the cytoplasm of the bacteriocyte to the haemolymph migrate to the embryo at the cellular blastoderm stage, through the opening at its posterior pole. Next, the bacteria enter the cytoplasm of newly formed bacteriocytes. The concept of the relationship between the geographic distribution of Hormaphidini aphids and the presence/absence of bacterium Buchnera aphidicola is discussed.     

Ultrastructure, distribution, and transovarial transmission of symbiotic microorganisms in Nysius ericae and Nithecus jacobaeae (Heteroptera: Lygaeidae: Orsillinae)

The organization of the symbiotic system (i.e., distribution and ultrastructure of symbionts) and the mode of inheritance of symbionts in two species, Nysius ericae and Nithecus jacobaeae belonging to Heteroptera: Lygaeidae, are described. Like most hemipterans, Nysius ericae and Nithecus jacobaeae harbor obligate prokaryotic symbionts. The symbiotic bacteria are harbored in large, specialized cells termed bacteriocytes which are localized in the close vicinity of the ovaries as well as inside the ovaries. The ovaries are composed of seven ovarioles of the telotrophic type. Bacteriocytes occur in each ovariole in the basal part of tropharium termed the infection zone. The bacteriocytes form a ring surrounding the early previtellogenic oocytes. The cytoplasm of the bacteriocytes is tightly packed with large elongated bacteria. In the bacteriocytes of Nysius ericae, small, rod-shaped bacteria also occur. Both types of bacteria are transovarially transmitted from one generation to the next.     

Independent origins and horizontal transfer of bacterial symbionts of aphids

Many insect groups have obligate associations with primary endosymbionts: mutualistic bacteria that are maternally transmitted and derived from an ancient infection. Often, the same insects are hosts to 'secondary' bacterial symbionts which are maternally transmitted but relatively labile within host lineages. To explore the dynamics of secondary symbiont associations in aphids, we characterized bacteria infecting 15 species of macrosiphine aphids using DNA sequencing, diagnostic polymerase chain reaction (PCR), diagnostic restriction digests, phylogenetic analyses, and electron microscopy to examine aphids from nature and from laboratory colonies. Three types of bacteria besides Buchnera were found repeatedly; all three fall within the Enterobacteriaceae. The R-type has a 16S rDNA less than 0.1% different from that of the secondary symbiont previously reported from Acyrthosiphon pisum and is related to Serratia species. The T-type includes a symbiont previously reported from a whitefly; the U-type comprises a new cluster near the T-type. The T-type was found in every one of 40 Uroleucon ambrosiae clones collected throughout the United States. In contrast, A. pisum individuals were infected by any combination of the three symbiont types. Secondary symbionts were maternally transmitted for 11 months within laboratory-reared A. pisum clones and were present in sexually produced eggs. PCR screens for a bacteriophage, APSE-1, indicated its presence in both A. pisum and U. ambrosiae containing secondary symbionts. Electron microscopy of R-type and T-type bacteria in A. pisum and in U. ambrosiae revealed rod-shaped organisms that attain extremely high densities within a few bacteriocytes.     

Symbiotic microorganisms in <Emphasis Type="Italic">Puto superbus</Emphasis> (Leonardi, 1907) (Insecta,Hemiptera, Coccomorpha: Putoidae)

The scale insect Puto superbus (Putoidae) lives in mutualistic symbiotic association with bacteria. Molecular phylogenetic analyses have revealed that symbionts of P. superbus belong to the gammaproteobacterial genus Sodalis. In the adult females, symbionts occur both in the bacteriocytes constituting compact bacteriomes and in individual bacteriocytes, which are dispersed among ovarioles. The bacteriocytes also house a few small, rod-shaped Wolbachia bacteria in addition to the numerous large, elongated Sodalis-allied bacteria. The symbiotic microorganisms are transovarially transmitted from generation to generation. In adult females which have choriogenic oocytes in the ovarioles, the bacteriocytes gather around the basal part of the tropharium. Next, the entire bacteriocytes pass through the follicular epithelium surrounding the neck region of the ovariole and enter the space between oocyte and follicular epithelium (perivitelline space). In the perivitelline space, the bacteriocytes assemble extracellularly in the deep depression of the oolemma at the anterior pole of the oocyte, forming a “symbiont ball”.     

Symbiosis in the green leafhopper,Cicadella viridis (Hemiptera,Cicadellidae). Association in statu nascendi?

The green leafhopper, Cicadella viridis lives in symbiotic association with microorganisms. The ultrastructural and molecular analyses have shown that in the body of the C. viridis two types of bacteriocyte endosymbionts are present. An amplification and sequencing of 16S rRNA genes revealed that large, pleomorphic bacteria display a high similarity (94–100%) to the endosymbiont ‘Candidatus Sulcia muelleri’ (phylum Bacteroidetes), whereas long, rod-shaped microorganisms are closely related to the γ-proteobacterial symbiont Sodalis (97–99% similarity). Both endosymbionts may be harbored in their own bacteriocytes as well as may co-reside in the same bacteriocytes. The ultrastructural observations have revealed that the Sodalis-like bacteria harboring the same bacteriocytes as bacterium Sulcia may invade the cells of the latter. Bacteria Sulcia and Sodalis-like endosymbionts are transovarially transmitted from one generation to the next. However, Sodalis-like endosymbionts do not invade the ovaries individually, but only inside Sulcia cells. Apart from bacteriocyte endosymbionts, in the body of C. viridis small, rod-shaped bacteria have been detected, and have been identified as being closely related to γ-proteobacterial microorganism Pectobacterium (98–99% similarity). The latter are present in the sheath cells of the bacteriomes containing bacterium Sulcia as well as in fat body cells.     

Differences in bacterial diversity of host-associated populations of Phylloxera notabilis Pergande (Hemiptera: Phylloxeridae) in pecan and water hickory

Host‐associated differentiation (HAD) is the presence of genetically divergent, host‐associated populations. It has been suggested that microbial symbionts of insect herbivores may play a role in HAD by allowing their insect hosts to use different plant species. The objective of this study was to document if host‐associated populations of Phylloxera notabilis Pergande (Hemiptera: Phylloxeridae) in pecan and water hickory corresponded with differences in the composition of their associated bacteria. To test this hypothesis, we characterized the symbionts present in P. notabilis associated with these two tree species through metagenomic analyses using 454 sequencing. Differences in bacterial diversity were found between P. notabilis populations associated with pecan and water hickory. The bacteria, Pantoea agglomerans and Serratia marcescens, were absent in the P. notabilis water hickory population, whereas both species accounted for more than 69.72% of bacterial abundance in the pecan population.     

Evidence of diversity and recombination in <Emphasis Type="Italic">Arsenophonus</Emphasis> symbionts of the <Emphasis Type="Italic">Bemisia tabaci</Emphasis>species complex

BACKGROUND: Maternally inherited bacterial symbionts infecting arthropods have major implications on host ecology and evolution. Among them, the genus Arsenophonus is particularly characterized by a large host spectrum and a wide range of symbiotic relationships (from mutualism to parasitism), making it a good model to study the evolution of host-symbiont associations. However, few data are available on the diversity and distribution of Arsenophonus within host lineages. Here, we propose a survey on Arsenophonus diversity in whitefly species (Hemiptera), in particular the Bemisia tabaci species complex. This polyphagous insect pest is composed of genetic groups that differ in many ecological aspects. They harbor specific bacterial communities, among them several lineages of Arsenophonus, enabling a study of the evolutionary history of these bacteria at a fine host taxonomic level, in association to host geographical range and ecology. RESULTS: Among 152 individuals, our analysis identified 19 allelic profiles and 6 phylogenetic groups, demonstrating this bacterium's high diversity. These groups, based on Arsenophonus phylogeny, correlated with B. tabaci genetic groups with two exceptions reflecting horizontal transfers. None of three genes analyzed provided evidence of intragenic recombination, but intergenic recombination events were detected. A mutation inducing a STOP codon on one gene in a strain infecting one B. tabaci genetic group was also found. Phylogenetic analyses of the three concatenated loci revealed the existence of two clades of Arsenophonus. One, composed of strains found in other Hemiptera, could be the ancestral clade in whiteflies. The other, which regroups strains found in Hymenoptera and Diptera, may have been acquired more recently by whiteflies through lateral transfers. CONCLUSIONS: This analysis of the genus Arsenophonus revealed a diversity within the B. tabaci species complex which resembles that reported on the larger scale of insect taxonomy. We also provide evidence for recombination events within the Arsenophonus genome and horizontal transmission of strains among insect taxa. This work provides further insight into the evolution of the Arsenophonus genome, the infection dynamics of this bacterium and its influence on its insect host's ecology.     

Actual distribution of bacteriocytes in the trophosome of a beard worm (Oligobrachia mashikoi, Siboglinidae, Annelida): clarification using whole-mount in situ hybridization

Beard worms (Siboglinidae, Polychaeta) lack a mouth and a digestive tract and harbour chaemosynthetic bacteria in the bacteriocytes of the trophosome. Since beard worms depend on the organic compounds produced by the bacteria for nourishment, the bacteriocytes should be efficient in exchanging various substances with body fluids. For this reason, it is important to determine how the bacteriocytes are organized in the trophosome. As the first step of the present study, the appearance of bacteriocytes was examined in routinely stained paraffin sections. Second, visualization of the actual distribution of the bacteriocytes was attempted using whole‐mount in situ hybridization with a probe of the 16S rRNA nucleotide sequence of the bacterium. After routine haematoxylin & eosin staining, the bacteriocytes appeared to be aligned in cell cords accompanied with nutrient‐deposit cells that extended from both sides of the trophosome toward the dorsal side and folded up in the coelomic spaces. In whole‐mount preparations, however, bacteriocytes with intense signals of 16S rRNA were seen three‐dimensionally as many irregular leaves arranged from both sides of the ventral vessel toward the dorsal vessel. We will discuss the physiological significance of this characteristic distribution of the bacteriocytes in the present species.     

Replication of the Endosymbiotic Bacterium Blochmannia floridanus Is Correlated with the Developmental and Reproductive Stages of Its Ant Host

The dynamics of replication of the intracellular endosymbiotic bacterium Blochmannia floridanus was determined during the larval development of its host ant Camponotus floridanus by real-time quantitative PCR. The bacteria were found to proliferate during pupation and immediately after the eclosion of the imagines (adult ants). In older workers the number of bacteria present in the midgut bacteriocytes decreased significantly. In contrast, the bacterial population in the ovaries was dependent on the reproductive state of the animal. An age-dependent degeneration of the midgut bacteriocytes was also investigated by microscopic techniques in males and female castes of the closely related ant species C. herculeanus and C. sericeiventris, respectively, with similar results and supports the concept of age-dependent degeneration of the midgut bacteriocytes in all castes.     

Co-cladogenesis spanning three phyla: leafhoppers (Insecta: Hemiptera: Cicadellidae) and their dual bacterial symbionts

Endosymbioses are a major form of biological complexity affecting the ecological and evolutionary diversification of many eukaryotic groups. These associations are exemplified by nutritional symbioses of insects for which phylogenetic studies have demonstrated numerous cases of long-term codiversification between a bacterial and a host lineage. Some insects, including most leafhoppers (Insecta: Hemiptera: Cicadellidae), have more than one bacterial symbiont within specialized host cells, raising questions regarding the patterns of codiversification of these multiple partners and the evolutionary persistence of complex symbiotic systems. Previous studies reported the presence of two dominant symbiont types in a member of the leafhopper subfamily Cicadellinae (sharpshooters). In this study, 16S rRNA sequences were obtained and used to examine the occurrence and evolutionary relationships of the two dominant symbiont types across 29 leafhopper species. Candidatus Sulcia muelleri (Bacteroidetes) was detected in all leafhopper species examined, a finding that is consistent with a previous report of its ancient association with the Auchenorrhyncha (a grouping that includes leafhoppers, treehoppers, cicadas, planthoppers, and spittlebugs). Baumannia cicadellinicola (Proteobacteria), previously known from only five sharpshooter species, was found only in the sharpshooter tribes Cicadellini and Proconiini, as well as in the subfamily Phereurhininae. Mitochondrial and nuclear gene sequences were obtained and used to reconstruct host phylogenies. Analyses of host and symbiont data sets support a congruent evolutionary history between sharpshooters, Sulcia and Baumannia and thus provide the first strong evidence for long-term co-inheritance of multiple symbionts during the diversification of a eukaryotic host. Sulcia shows a fivefold lower rate of 16S rDNA sequence divergence than does Baumannia for the same host pairs. The term 'coprimary' symbiont is proposed for such cases.     

斑透翅蝉唾液腺细菌群落组成研究

【目的】为明晰蝉类昆虫唾液腺中细菌的组成及其中是否存在内共生菌Candidatus Sulcia muelleri。【方法】以斑透翅蝉Hyalessa maculaticollis (Motschulsky)为材料,采用16S rRNA限制性内切酶片段长度多态性(RFLP)对其唾液腺细菌群落组成进行分析。【结果】斑透翅蝉唾液腺中共存在7种细菌,分别属于变形菌门和厚壁菌门;其中绿脓假单胞杆菌Pseudomonas aeruginosa和肠杆菌Enterobacter sp.为优势细菌,分别占克隆总数的48.7%;另外5种细菌(反硝化细菌热单胞菌Thermomonas brevis、鞘胺醇单胞菌Sphingomonas sp.、芽孢杆菌Bacillus sp.、厌氧球菌Anaerococcus sp.和Methyloversatilis sp.)总共占克隆文库的2.05%。【结论】首次采用分子生物学方法明晰斑透翅蝉唾液腺的细菌群落;其细菌群落组成相对简单,且两种细菌占主导地位;此外,头喙亚目昆虫体内特有的内共生菌Candidatus Sulcia muelleri未在斑透翅蝉唾液腺中检测出,表明该共细菌可能仅在腹部的贮菌体中分布;斑透翅蝉唾液腺中的细菌是否普遍存在于蝉科昆虫唾液腺中以及在取食韧皮部汁液过程中的功能有待进一步研究。     

When mutualists are pathogens: an experimental study of the symbioses between Steinernema (entomopathogenic nematodes) and Xenorhabdus (bacteria)

In this paper, we investigate the level of specialization of the symbiotic association between an entomopathogenic nematode (Steinernema carpocapsae) and its mutualistic native bacterium (Xenorhabdus nematophila). We made experimental combinations on an insect host where nematodes were associated with non-native symbionts belonging to the same species as the native symbiont, to the same genus or even to a different genus of bacteria. All non-native strains are mutualistically associated with congeneric entomopathogenic nematode species in nature. We show that some of the non-native bacterial strains are pathogenic for S. carpocapsae. When the phylogenetic relationships between the bacterial strains was evaluated, we found a clear negative correlation between the effect a bacterium has on nematode fitness and its phylogenetic distance to the native bacteria of this nematode. Moreover, only symbionts that were phylogenetically closely related to the native bacterial strain were transmitted. These results suggest that co-evolution between the partners has led to a high level of specialization in this mutualism, which effectively prevents horizontal transmission. The pathogenicity of some non-native bacterial strains against S. carpocapsae could result from the incapacity of the nematode to resist specific virulence factors produced by these bacteria.     

Phylogenetic Placement of Pentatomid Stink Bug Gut Symbionts

Insect bacterial symbionts are ubiquitous, however, only a few groups of host families have been well studied in relation to their associations with microbes. The determination of the phylogenetic relationships among bacteria associated with different species within an insect family can provide insights into the biology and evolution of these interactions. We studied the phylogenetic placement of vertically transmitted bacterial symbionts associated with the posterior midgut (crypt-bearing) region of pentatomid stink bugs (Hemiptera, Pentatomidae). Our results demonstrate that different host species carried one major bacterium in their midgut. Phylogenetic analyses of the 16S rRNA gene sequences obtained from the midgut of stink bugs placed all symbionts in a clade with Erwinia and Pantoea species, both plant-associated bacteria. Results indicate that symbiont monophyly occurs among recently diverged taxa (e.g., within a genus) but does not occur in the Pentatomidae. Results suggest that these vertically transmitted symbionts are occasionally replaced by other taxonomically similar bacteria over evolutionary time. Our findings highlight how the evolutionary history of hemipteran symbionts in unexplored host families may have unpredictable levels of complexity.     

Histochemical demonstration of oxidoreductase activities in the fat body and symbionts of Blattella germanica (Blattodea) following chlortetracycline-treatment

The metabolic apport of prokaryotic symbionts in the fat body of Blattella germanica was investigated by histoenzymatic methods, using chlortetracycline-treated and normal strains. In the experimental insects, bacteriocytes showed a decreased oxidoreductase activity, whereas the staining intensity of the other cell types was generally unchanged. Electron microscopic observations showed that some bacteria were still present in the bacteriocytes of the treated insects, but exhibited degeneration patterns to a different extent; therefore, they are not likely to carry on any enzymatic activity. Hence, chlortetracycline, an antibiotic that blocks the transovaric transmission of the symbionts, is active also on the endocellular symbionts of the fat body.     

Two ancient bacterial endosymbionts have coevolved with the planthoppers (Insecta: Hemiptera: Fulgoroidea)

ABSTRACT: BACKGROUND: Members of the hemipteran suborder Auchenorrhyncha (commonly known as planthoppers, tree- and leafhoppers, spittlebugs, and cicadas) are unusual among insects known to harbor endosymbiotic bacteria in that they are associated with diverse assemblages of bacterial endosymbionts. Early light microscopic surveys of species representing the two major lineages of Auchenorrhyncha (the planthopper superfamily Fulgoroidea; and Cicadomorpha, comprising Membracoidea [tree- and leafhoppers], Cercopoidea [spittlebugs], and Cicadoidea [cicadas]), found that most examined species harbored at least two morphologically distinct bacterial endosymbionts, and some harbored as many as six. Recent investigations using molecular techniques have identified multiple obligate bacterial endosymbionts in Cicadomorpha; however, much less is known about endosymbionts of Fulgoroidea. In this study, we present the initial findings of an ongoing PCR-based survey (sequencing 16S rDNA) of planthopper-associated bacteria to document endosymbionts with a long-term history of codiversification with their fulgoroid hosts. RESULTS: Results of PCR surveys and phylogenetic analyses of 16S rDNA recovered a monophyletic clade of Betaproteobacteria associated with planthoppers; this clade included Vidania fulgoroideae, a recently described bacterium identified in exemplars of the planthopper family Cixiidae. We surveyed 77 planthopper species representing 18 fulgoroid families, and detected Vidania in 40 species (representing 13 families). Further, we detected the Sulcia endosymbiont (identified as an obligate endosymbiont of Auchenorrhyncha in previous studies) in 30 of the 40 species harboring Vidania. Concordance of the Vidania phylogeny with the phylogeny of the planthopper hosts (reconstructed based on sequence data from five genes generated from the same insect specimens from which the bacterial sequences were obtained) was supported by statistical tests of codiversification. Codiversification tests also supported concordance of the Sulcia phylogeny with the phylogeny of the planthopper hosts, as well as concordance of planthopper-associated Vidania and Sulcia phylogenies. CONCLUSIONS: Our results indicate that the Betaproteobacterium Vidania is an ancient endosymbiont that infected the common ancestor of Fulgoroidea at least 130 million years ago. Comparison of our findings with the early light-microscopic surveys conducted by Muller suggests that Vidania is Muller's x-symbiont, which he hypothesized to have codiversified with most lineages of planthoppers and with the Sulcia endosymbiont.