Ultrastructure of the bacteriome and bacterial symbionts in the Asian citrus psyllid, Diaphorina citri

ABSTRACT The Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) is a notorious agricultural pest that transmits “Candidatus Liberibacter” spp. (Alphaproteobacteria: Rhizobiales), the pathogens of the destructive citrus disease huanglongbing. D. citri possesses a specialized organ called the bacteriome, consisting of uninucleate bacteriocytes and a syncytium. The bacteriocytes harbor “Candidatus Carsonella ruddii” (Gammaproteobacteria: Oceanospirillales), a nutritional symbiont that provides essential amino acids scarce in the phloem sap diet. The syncytium contains “Candidatus Profftella armatura” (Gammaproteobacteria: Burkholderiales), a defensive symbiont synthesizing diaphorin, a polyketide inhibitory to various organisms. Besides these obligate mutualists, Wolbachia (Alphaproteobacteria: Rickettsiales), a facultative symbiont, is also widespread in D. citri. To understand the morphological features of the bacteriome and symbionts, this study analyzed their ultrastructure using transmission electron microscopy. Putative Wolbachia cells were observed in the uninucleate bacteriocytes, which were packed with numerous Carsonella cells surrounded by host mitochondria, rough endoplasmic reticula, and the Golgi apparatus. Carsonella contained many ribosomes, parts of which formed conspicuous aggregates. The Carsonella envelope was lined with fiber bundles forming a net-like structure. In Profftella, numerous tubular structures were observed, each approximately 300 nm in diameter, consisting of several intertwined fibers. Some Profftella cells exhibited surface protrusions. The syncytium occasionally contained Profftella cells, apparently in the degradation process. Host organelles were less abundant in the syncytium than in the uninucleate bacteriocytes. Mitochondria in the syncytium had less developed cristae than those in the uninucleate bacteriocytes. These observations established a basis for understanding interactions among the host cells and bacterial symbionts. IMPORTANCE Omics analyses suggested a mutually indispensable tripartite association among the host D. citri and organelle-like bacteriome associates, Carsonella and Profftella, which are vertically transmitted through host generations. This relationship is based on the metabolic complementarity among these organisms, which is partly enabled by horizontal gene transfer between partners. However, little was known about the fine morphology of the symbionts and the bacteriome, the interface among these organisms. As a first step to address this issue, the present study performed transmission electron microscopy, which revealed previously unrecognized ultrastructures, including aggregations of ribosomes in Carsonella, numerous tubes and occasional protrusions of Profftella, apparently degrading Profftella, and host organelles with different abundance and morphology in distinct cell types. These findings provide insights into the behaviors of the symbionts and host cells to maintain the symbiotic relationship in D. citri.

V arious insect lineages possess a specialized organ called the bacteriome to harbor microbial symbionts (1)(2)(3).These insects are mainly exemplified by taxa that feed only on nutrient-deficient diets, including plant sap and vertebrate blood, and thus include numerous agricultural and medical pests.Although not only the phylogeny of symbionts but also the morphology, localization, and developmental origin of the bacteriome vary depending on insect lineages, bacteriome-associated symbionts tend to converge to organelle-like mutualists that provide essential nutrients for host survival (1)(2)(3).The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Psylloidea: Psyllidae: Diaphorininae), is a notorious agricultural pest that transmits "Candidatus Liberibacter" spp.(Alphaproteobacteria: Rhizobiales, hereafter Liberibacter), the pathogens of a destructive and incurable citrus disease known as huanglongbing or greening disease (4)(5)(6).While association with Liberibacter is transient, D. citri has more intimate and evolutionarily stable relationships with bacteriome-associated microbes that are transovarially transmitted through host generations (7)(8)(9).The abdominal hemocoel of D. citri, as in other psyllid species (10)(11)(12)(13)(14)(15)(16), contains a large yellow bilobed bacteriome that consists of numerous uninucleate bacteriocytes, a central syncytium surrounded by bacteriocytes, and an envelope to encase the whole organ (7)(8)(9).The uninucleate bacteriocytes harbor "Candidatus Carsonella ruddii" (Gammaproteobacte ria: Oceanospirillales, hereafter Carsonella) (17), a typical nutritional symbiont, which provides the host psyllid with essential amino acids (8,(18)(19)(20)(21) that are scarce in the phloem sap diet (22,23).Carsonella is referred to as the "primary symbiont" because it is conserved among host species and is thus considered essential in Psylloidea (8,17,18,21,(24)(25)(26)(27)(28)(29).The syncytium of the psyllid bacteriome usually contains another bacterial lineage categorized as a "secondary symbiont, " which is phylogenetically diverse among psyllid lineages, suggesting its repeated replacements during the evolution of Psylloidea (19,(24)(25)(26)(27)(28)(29)(30)(31).The secondary symbiont housed in the D. citri bacteriome is "Candidatus Profftella armatura" (Gammaproteobacteria: Burkholderiales, hereafter Profftella) (8,9,20,26), a unique organelle-like defensive symbiont, whose primary role appears to protect the holobiont (host psyllid and bacteriome-associated mutualists) from natural enemies, using diaphorin, a polyketide inhibitory to various organisms (8,20,(32)(33)(34)(35).Omics analyses indicate a mutually indispensable tripartite association among the host D. citri and these symbionts.During their evolution as obligate mutualists confined in the bacteriome, the genomes of Carsonella and Profftella have been drastically reduced to 170 and 460 kb, respectively (8).Carsonella lacks numerous genes for biological processes apparently essential for survival but retains many genes for the biosynthesis of essential amino acids, which is consistent with the presumed role of Carsonella to supplement nutrition (8,(18)(19)(20)(21).In contrast, Profftella has few genes for nutrient production, but 15% of the genome is devoted to gene clusters for the biosynthe sis of diaphorin, a defensive secondary metabolite.The metabolic genes encoded in the Profftella genome are largely nonredundant with those of Carsonella, indicating complementarity between these symbionts (8).Furthermore, many host genes are upregulated in the bacteriome to complement the incomplete biosynthetic pathways deduced from the highly reduced Carsonella genome (16,36).The majority of them are native animal genes, but some are shown to be of bacterial origin, which the host psyllid has horizontally acquired.Most notable are two divergent gene copies encoding argininosuccinate lyases that are responsible for the terminal step in the arginine biosynthesis pathway.Phylogenetic analyses strongly suggest that these genes are directly transferred from Carsonella (16).An ortholog is retained in the Carsonella genome but appears to have acquired a novel function independent of arginine biosynthesis.Another notable example is a gene (ribC) involved in the biosynthesis of riboflavin or vitamin B 2 (16,36), which is also scarce in the phloem sap diet (22).Although Profftella lacks most genes for nutritional provisioning, it exceptionally retains all genes for the riboflavin biosynthetic pathway other than ribC (8), suggesting metabolic complementarity between Profftella and the host psyllid (16).The donor bacterium of the psyllid ribC is uncertain but appears neither Profftella nor Carsonella.
Although these horizontally acquired genes were first discovered in Pachypsylla venusta (Psylloidea: Carsidaridae), screening of genomic/transcriptomic data of D. citri and the potato psyllid Bactericera cockerelli (Triozidae) identified at least nine genes of bacterial origin shared by the three divergent psyllid species, indicating that the gene transfer events have occurred before the radiation of the major psyllid lineages (16).The mutually indispensable tripartite association based on genetic complementarity among the host and symbionts should depend on coordinated metabolic processes.However, little is known about how and which compounds (e.g., metabolites, proteins, or RNAs) are transferred among the host cytoplasm and symbionts.

Insects
An established colony of D. citri, originally collected from the island of Amami Oshima, Kagoshima, Japan, was maintained on the saplings of the orange jasmine, Murraya paniculata (Rutaceae), individually covered with insect-rearing sleeves (L70 cm × W30 cm, Bugdorm Store; Taichung, Taiwan).The plant pots were kept in incubators at 28°C with a 16:8 (light:dark)-h photoperiod.Our previous microbiome analysis revealed that this colony is free from Liberibacter but is infected with Wolbachia of supergroup B, besides Carsonella and Profftella (26).Adults 5-10 days after emergence were collected from M. paniculata saplings using an insect aspirator and then caged in a plastic dish on ice for 5 min to immobilize them.They were sexed under a stereomicroscope, and bacteriomes were dissected from adult females of D. citri in phosphate-buffered saline.

Transmission electron microscopy
The dissected bacteriomes were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M sodium phosphate buffer [0.1 M NaH 2 PO 4 and 0.1 M Na 2 HPO 4 (pH 7.4)] for approximately 24 h.After rinsing with 0.05 M potassium phosphate buffer [0.05 M KH 2 PO 4 and 0.05 M Na 2 HPO 4 (pH 7.4)], the specimens were postfixed with 2% osmium tetroxide in 0.05 M potassium phosphate buffer at 4°C for 2 h.After rinsing with 0.05 M potassium phosphate buffer, the fixed specimens were dehydrated in a graded series of ethanol (30%, 50%, 70%, and 90% once, and with 100% thrice, 15 min for each step).Subsequently, the specimens were treated with propylene oxide thrice for 10 min to replace ethanol, which were then embedded in Quetol 651 epoxy resin.The resin was polymerized at 60°C for 24 h.Ultrathin (80-90 nm thick) sections were obtained by ultramicrotomy and stained with 2% uranyl acetate and modified Sato's lead solution [1.0 g each of Pb(NO 3 ) 2 -3H 2 O, Pb(CH 3 COO) 2 -3H 2 O, and 2.0 g of Na 3 C 6 H 5 O 7 -2H 2 O dissolved in 82 mL of distilled water and 18 mL of 4% NaOH aqueous solution] (69).TEM observation was performed using a JEOL JEM-1200EX electron microscope or Hitachi H-7600.

Overview
The overall structure of the bacteriome of D. citri was as previously described (7)(8)(9).It consisted of numerous uninucleate bacteriocytes harboring Carsonella, a central syncytium harboring Profftella, and an envelope enclosing the whole organ (Fig. 1A).Some tracheoles were observed in the syncytium, indicating that this region is aerated (Fig. 1B).Although most uninucleate bacteriocytes were located peripherally to surround the central syncytium (Fig. 1A), the cells of the same type were occasionally observed within the syncytium (arrows in Fig. 1C and D).Moreover, a few structures resembling Carsonella in the bacteriocyte (Fig. 1E) were also observed in the syncytial cytoplasm (arrowheads in Fig. 1A, C, and F).As previous FISH analyses did not detect clear signals of free Carsonella cells in the syncytium (8,9), further studies are required to determine whether these structures are truly Carsonella.Relatively small numbers of putative Wolbachia cells were observed in the uninucleate bacteriocytes (Fig. 1G through I) but not in the syncytium.The putative Wolbachia cells appeared mostly spherical, with a diameter of approximately 600 nm, much smaller than Carsonella and Profftella.They were comparable in size to the transverse sections of mitochondria but easily distinguishable as they lacked crista (Fig. 1H and I).The morphology of these cells was highly similar to that of TEM images of Wolbachia observed in various cell types of diverse host organisms, including the bacteriocyte of the whitefly Bemisia tabaci (Hemiptera: Sternorrhyncha: Aleyrodoidea: Aleyrodidae), the phloem of the cotton plant Gossypium hirsutum (Malvales: Malvaceae) that B. tabaci feeds on (70), ovary cells of the mosquito Aedes aegypti (Diptera: Culicidae) (71), cell lines of the mosquitos A. aegypti and Anopheles gambiae (Diptera: Culicidae) (72), and the fruit fly Drosophila melanogaster (Diptera: Drosophilidae) (73), and hindgut cells and midgut caeca of the woodlouse Armadillidium vulgare (Crustacea: Malacostraca: Isopoda) (74).Some of the putative Wolbachia cells in D. citri bacteriocytes showed a gap between the double-layered cell envelope and the outermost structure, which appeared to be the host membrane encasing Wolbachia (Fig. 1I).

Carsonella and uninucleate bacteriocytes
Numerous sections of Carsonella were observed in the uninucleate bacteriocyte (Fig. 2A), which was consistent with previous DNA staining and FISH observations, showing that uninucleate bacteriocytes are packed with pleiomorphic, mostly tubular, Carsonella cells (9,18,21).The uninucleate bacteriocytes were rich in mitochondria ( Fig. 2A, B, E, and F), endoplasmic reticula with ribosomes (rough endoplasmic reticula, Fig. 2B through F), and the Golgi apparatus (Fig. 2D and E).Many mitochondria (Fig. 2A, B, E, and F) and rough endoplasmic reticula (Fig. 2B through F) were observed surrounding and in contact with Carsonella cells, implying close associations between Carsonella and these host organelles.Although less abundant, Golgi apparatuses were likewise observed in close proximity to Carsonella (Fig. 2D and E).While it is thought that Carsonella is enveloped in a triple-layered membrane (12,13), the ultrastructure of the Carsonella envelope was not clearly observed in this study.However, fiber bundles were observed just under the envelope of Carsonella (Fig. 2C).These structures appeared to correspond to the "peripheral macula" previously reported for Carsonella of other psyllid species, Cacopsylla pyricola (Psyllidae: Psyllinae) (12) and Anomoneura mori (Psyllidae: Psyllinae) (13).The fiber bundles were arranged both longitudinally and transversely, suggesting to form a net-like structure lining the envelope of Carsonella.Carsonella cells were highly electron dense, and numerous darker spots were observed therein (Fig. 1 and 2).Carsonella contained a large number of ribosomes, which may partly account for the high electron density (Fig. 2B through D).The dark spots that previous studies referred to as "unidentified electron-dense aggregates" (17) or "central granular masses" (13) appeared to be aggregations of ribosomes (Fig. 2C and D).There was a remarkable variation in electron density among Carsonella cells, even within a single bacteriocyte (Fig. 1A, 2E and F).We currently have no idea what accounts for this variation.

Profftella and syncytium
Numerous sections of Profftella with various shapes were observed in the syncytium (Fig. 3A through I), suggesting that Profftella are pleiomorphic.The Profftella cells were much less electron dense than Carsonella (Fig. 1 to 3).Conspicuous components found in Profftella cells were tubular structures sectioned both transversely (arrows in Fig. 3A through D) and longitudinally (arrowheads in Fig. 3A through D).These structures were approximately 300 nm in diameter, consisting of several intertwined fibers (Fig. 3C and  D).It appeared that a single Profftella cell can contain multiple tubes.Although ribo somes were less abundant in Profftella than in Carsonella, many appeared to colocalize with the tubes (Fig. 3C and D).Profftella cells were observed to be encased in a threelayered envelope, the outermost of which probably corresponds to a host-derived membrane (Fig. 3F).Some Profftella cells exhibited protrusions on the surface (asterisks in Fig. 3A, B, F, and G).The syncytium occasionally contained structures that appeared to be Profftella cells in the degradation process (Fig. 3G through I).Host organelles, including mitochondria and endoplasmic reticula with ribosomes, were observed in the syncytium, but they were less abundant than in the uninucleate bacteriocytes (Fig. 3A through I).Furthermore, mitochondria in the syncytium had fewer cristae than those in the uninucleate bacteriocytes (Fig. 3E).

DISCUSSION
This study revealed the ultrastructure of the bacteriome and associated symbionts in D. citri.The overall structure of the bacteriome was as previously described, in which most uninucleate bacteriocytes were located peripherally to surround the central syncytium (Fig. 1A).However, some uninucleate bacteriocytes were situated within the syncytium (Fig. 1C and D), which may facilitate the transport of compounds, potentially including metabolites, proteins, or RNAs, between Carsonella and Profftella.Furthermore, a few structures resembling Carsonella were also observed in the syncytial cytoplasm (Fig. 1A,  C and F).Although previous FISH analysis detected freed Carsonella cells in the hemocoel of D. citri (9), which were likely in the process of transfer from the bacteriome to the ovary where the symbionts infect oocytes, no such cells were clearly observed in the syncy tium.If the structures found in the present study are truly Carsonella, this proximity to Profftella may further facilitate material transfer between these symbionts.However, because the environmental conditions appear to differ between the uninucleate bacteriocyte and the syncytium, as suggested by the distinct abundance and morphol ogy of host organelles (Fig. 2 and 3), the syncytium may not be a suitable habitat for Carsonella.Further studies are required to determine whether these structures are truly Carsonella.Besides, limited numbers of Wolbachia cells were observed in the uninucleate bacteriocytes (Fig. 1G, H and I), which potentially allow Wolbachia to enjoy the environ ment of the bacteriocyte, where various metabolites are assumed to be actively synthe sized and transferred among the host and symbionts (16,36).However, previous analyses FIG 1 (Continued) area in the rectangle in G.The putative Wolbachia cell is comparable in size to the transverse sections of mitochondria but lacks crista.(I) A putative Wolbachia cell with a gap between its double-layered cell envelope and the outermost structure, which appears to be the host membrane encasing Wolbachia.be, bacteriome envelope; c, Carsonella; m, mitochondrion; n, host nucleus; p, Profftella; sy, syncytium; t, tracheole; ubp, uninucleate bacteriocyte at the peripheral region; ubs, uninucleate bacteriocyte in the syncytium; w, putative Wolbachia.(E and F) Other images of peripheral uninucleate bacteriocytes surrounding the syncytium.Carsonella cells show a close proximity with host organelles, including mitochondria, endoplasmic reticula with ribosomes, and the Golgi apparatus.A variation in electron density among Carsonella cells is observed.c, Carsonella; er, endoplasmic reticulum with ribosomes; fbl, fiber bundle arranged longitudinally; fbt, fiber bundle arranged transversely; ga, Golgi apparatus; m, mitochondrion; n, host nucleus; p, Profftella; ra, ribosomal aggregate; sy, syncytium; ubp, uninucleate bacteriocyte at the peripheral region.showed that the relative abundance of Wolbachia was much higher in Malpighian tubules and the gut (43), implying that the bacteriome is not the best habitat for Wolbachia.
The uninucleate bacteriocytes were rich in host organelles including mitochondria, rough endoplasmic reticula, and the Golgi apparatus, many of which were observed surrounding and in contact with Carsonella cells (Fig. 2).This arrangement potentially facilitates the delivery of proteins and metabolites required to maintain the bacteriocyte symbiosis, being consistent with metabolic complementarity between the host and Carsonella, which was suggested by genomic and transcriptomic analyses (8,16,(18)(19)(20)36).In the case of aphids, sternorrhynchan insects closely related to psyllids, proteins synthesized by the host bacteriocytes are shown to be transported to the endosymbiont (62).It would be interesting to assess if similar integrated systems have evolved in Psylloidea.The close proximity between Carsonella and host organelles is reminiscent of the case of the protist Paramecium bursaria.Intimate connections were identified between the P. bursaria mitochondria and symbiotic algae via the host membrane that contains algae, implying the transfer of proteins and essential metabolites (75).In P. bursaria, only cryo-fixation identified direct contacts of the vacuole membrane to the mitochondrial membrane and the cell wall of the symbiotic algae, which was unattained by the chemical fixation.Therefore, cryo-electron microscopy may reveal finer structures showing more intimate host-symbiont interactions in the bacteriome symbiotic system of D. citri.
This study identified fiber bundles just under the envelope of Carsonella (Fig. 2C).The fibers were arranged both longitudinally and transversely, suggesting to form a net-like structure lining the Carsonella envelope.These structures appeared to correspond to the "peripheral macula" previously reported for Carsonella of other psyllid species, C. pyricola (Psyllidae: Psyllinae) (12) and A. mori (Psyllidae: Psyllinae) (13).However, to the best of our knowledge, similar structures have not been observed in any other bacteria, including other bacteriome-associated symbionts in insects (76)(77)(78)(79)(80)(81)(82).As Carsonella cells are relatively large and can be extremely long (9,18,21), these linings may play an important role in providing mechanical support to the cell structure of Carsonella.Within Carsonella cells, numerous ribosomes were observed, parts of which formed conspicu ous aggregations (Fig. 2C and D) reminiscent of the nucleolus, the site of ribosome biogenesis in eukaryotes (83).A previous study showed that Carsonella stained with DAPI exhibits numerous spots with higher signal intensities within the cell (21).The aggregates observed in the present study may correspond to these signal spots, which potentially contain higher amounts of nucleic acids.Further studies determining the precise localization of genomic copies, ribosomes, and their precursors in Carsonella would provide clues to understand the mechanisms for organizing numerous copies (21) of the drastically reduced genome (8,(18)(19)(20) and expressing genes encoded therein.
A characteristic feature of Profftella cells revealed in this study is the presence of tubular structures, which are approximately 300 nm in diameter, consisting of several intertwined fibers (Fig. 3A through D).Our previous metagenomic analysis did not detect DNA sequences corresponding to known viruses or bacteria other than Carsonella, Profftella, or Wolbachia (8), suggesting that the tubes are not parasitic microbes residing in Profftella.A large part of ribosomes appeared to colocalize with the tubes (Fig. 3C  and D), implying the involvement of these structures in the gene expression of Profftella.

FIG 3 (Continued)
Carsonella and the syncytium harboring Profftella.Mitochondria in the syncytium appear to have less developed cristae than their uninucleate bacteriocyte counterparts.(F) Three-layered envelopes that encase the Profftella cells.Protrusions (asterisks) are occasionally observed.(G) A structure that appears to be in the decomposition process (dagger), which was observed in the syncytium.The asterisk indicates a protrusion from Profftella.(H) and (I) Structures that appear to be Profftella cells in the decomposition process.c, Carsonella; er, endoplasmic reticulum with ribosomes; m, mitochondrion; n, host nucleus; p, Profftella; sy, syncytium; tsl, tubular structure sectioned longitudinally; tst, tubular structure sectioned transversely; ubp, uninucleate bacteriocyte at the peripheral region.
Further studies are required to determine the finer structures and functions of the tubes.This study showed that a three-layered envelope encapsulates Profftella cells, with the outermost layer probably derived from the host (Fig. 3F).The encasement of symbionts in a host membrane is a typical feature of various intracellular symbiotic systems (75)(76)(77)(78)(79)(80)(81)(82), which would facilitate the host's control over the symbionts.Apparent cell protru sions were observed on the surface of some Profftella cells (Fig. 3A, B, F, and G).These structures may facilitate interactions with the surrounding environment, including host organelles and cytoplasm of the syncytium, and other Profftella cells.Besides, structures that appeared to be degrading Profftella were occasionally observed in the syncytium (Fig. 3G through I).The degradation of Profftella would yield an opportunity to release the genomic DNA of Profftella into the cytoplasm of the syncytium.Although this study utilized D. citri uninfected with "Candidatus Liberibacter" spp., a previous study detected a considerable abundance of "Candidatus Liberibacter asiaticus" in the bacteriome of infected D. citri (43).Thus, the freed Profftella genome can potentially be a source of horizontal acquisition by the Liberibacter lineages, as exemplified in a transporter gene preserved in the Liberibacter lineages except for the most basal species L. crescens (64).
Host organelles, including mitochondria and endoplasmic reticula with ribosomes, were less abundant in the syncytium than in the uninucleate bacteriocytes (Fig. 3A through I).Moreover, mitochondria in the syncytium had fewer cristae than those in the uninucleate bacteriocytes (Fig. 3E).These observations imply that the syncytium is metabolically less active in comparison to bacteriocytes.

Conclusions
This study revealed ultrastructures of the D. citri bacteriome, including the following: (i) fiber bundles forming a net-like structure lining the Carsonella envelope, which may be essential to support the large and long Carsonella cells, (ii) aggregates of ribosomes in Carsonella, which are reminiscent of the eukaryotic nucleolus, (iii) many host organelles in contact with Carsonella cells in the uninucleate bacteriocyte, which may facilitate the delivery of proteins and metabolites required to maintain the bacteriocyte symbiosis, (iv) numerous tubes consisting of several intertwined fibers in Profftella, the function of which is uncertain, (v) protrusions on the surface of Profftella, which may facili tate interactions with the host cell components and other Profftella cells, (vi) degrad ing Profftella in the syncytium, providing opportunities for horizontal gene transfer between symbionts, and (vii) less abundant organelles, including mitochondria with less developed cristae, in the syncytium than in the uninucleate bacteriocytes.These observations established a basis for understanding interactions among the host cells and bacterial symbionts in D. citri.

FIG 1
FIG 1 Transmission electron micrographs showing an overview of the bacteriome in adult females of D. citri.(A) The basic structure of the D. citri bacteriome.Uninucleate bacteriocytes harboring Carsonella surround the central syncytium harboring Profftella.Limited numbers of Carsonella-like structures (arrowheads) are observed in the syncytium.(B) Tracheole observed in the syncytium.(C) Uninucleate bacteriocytes (arrows) and Carsonella-like structures (arrowheads) observed in the syncytium.(D) Another example of the uninucleate bacteriocyte observed in the syncytium.(E) Enlarged image of the area in the upper rectangle in C. Carsonella cells are observed in a peripheral uninucleate bacteriocyte surrounding the syncytium.(F) Enlarged image of the area in the lower rectangle in C. Carsonella-like structures are observed along with Profftella in the syncytium.(G) A peripheral uninucleate bacteriocyte containing a structure that appears to be Wolbachia.(H) Enlarged image of the (Continued on next page)

FIG 2
FIG 2 Ultrastructure of the uninucleate bacteriocyte and Carsonella.(A) Peripheral uninucleate bacteriocyte surrounding the syncytium.(B) Enlarged image of the area in the lower rectangle in A. Carsonella cells are adjacent to mitochondria and endoplasmic reticula with ribosomes.(C) Enlarged image of the area in the upper rectangle in A. Carsonella cells contain numerous ribosomes, some of which form conspicuous aggregates.Fiber bundles are observed near the surface of Carsonella cells.(D) Carsonella, a Golgi apparatus, and endoplasmic reticula with ribosomes adjacent to one another.

FIG 3
FIG 3 Ultrastructure of the syncytium and Profftella.(A) Syncytium containing Profftella, along with cell organelles including mitochondria.Tubular structures are observed in Profftella.Arrows and arrowheads indicate tubular structures sectioned transversely and longitudinally, respectively.Protrusions (asterisks) are observed in several Profftella cells.(B) Profftella cells containing tubular structures.Arrows and arrowheads indicate tubular structures arranged transversely and longitudinally, respectively.The asterisk indicates a protrusion from Profftella.Mitochondria and endoplasmic reticula with ribosomes are observed around Profftella cells.(C) Tubular structures observed in Profftella cells.Arrows and arrowheads indicate tubular structures sectioned transversely and longitudinally, respectively.(D) Tubular structures arranged longitudinally in Profftella cells.The structures appear to consist of intertwined fibers.(E) The interface of a uninucleate bacteriocyte harboring (Continued on next page)