Behavior of bacteriome symbionts during transovarial transmission and development of the Asian citrus psyllid

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a serious pest worldwide, transmitting Candidatus Liberibacter spp. (Alphaproteobacteria), the causative agents of a devastating citrus disease known as huanglongbing or greening disease. In a symbiotic organ called the bacteriome, D. citri possesses an organelle-like defensive symbiont, Candidatus Profftella armatura (Betaproteobacteria), and a nutritional symbiont, Ca. Carsonella ruddii (Gammaproteobacteria). Drastically reduced symbiont genomes and metabolic complementarity among the symbionts and D. citri indicate their mutually indispensable association. Moreover, horizontal gene transfer between the Profftella and Liberibacter lineages suggests ecological and evolutionary interactions between the bacteriome symbiont and the HLB pathogen. Using fluorescence in situ hybridization, we examined the behavior of Profftella and Carsonella during transovarial transmission and the development of D. citri. In the bacteriomes of sexually-mature female adults, symbionts transformed from an extremely elongated tubular form into spherical or short-rod forms, which migrated toward the ovary. The symbionts then formed mosaic masses, which entered at the posterior pole of the vitellogenic oocytes. After anatrepsis, Carsonella and Profftella migrated to the central and peripheral parts of the mass, respectively. Following the appearance of host nuclei, the mass cellularized, segregating Carsonella and Profftella in the central syncytium and peripheral uninucleate bacteriocytes, respectively. Subsequently, the uninucleate bacteriocytes harboring Profftella assembled at the posterior pole, while the syncytium, containing Carsonella, sat on the anterior side facing the germ band initiating katatrepsis. During dorsal closure, the syncytium was divided into uninuclear bacteriocytes, which surrounded the mass of bacteriocytes containing Profftella. Once fully surrounded, the bacteriocyte mass containing Profftella was fused into a syncytium. Prior to hatching, a pair of wing-like protrusions arose from both lateral sides of the bacteriome, which continued to grow throughout the nymphal stages. These findings provide a foundation for better understanding the intricate relationship between D. citri and its microbiota.


Introduction
The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Psylloidea: Liviidae) is an important agricultural pest that transmits Candidatus Liberibacter spp. (Alphaproteobacteria), the causative agents of a devastating citrus disease known as huanglongbing (HLB) or greening disease. All commercial citrus cultivars are susceptible to HLB, and a long latent period following infection allows rapid spread of the disease worldwide. Because HLB is currently incurable, controlling the D. citri vector is presently the most crucial part of HLB management [1].
D. citri possesses a symbiotic organ called the bacteriome in its hemocoel. The bacteriome harbors two distinct species of vertically transmitted symbionts: Candidatus Carsonella ruddii (Gammaproteobacteria) and Ca. Profftella armatura (Betaproteobacteria) [2]. Carsonella is a typical nutritional symbiont, providing its host with essential amino acids that are scarce in the diet of phloem sap. In contrast, Profftella appears to be an organelle-like defensive symbiont, producing toxins that protect the host from natural enemies. A mutually indispensable tripartite association among D. citri and the two symbionts is strongly suggested by the drastically reduced symbiont genomes, and by the metabolic complementarity among the organisms [2]. These features are partly the result of horizontal gene transfer between partners [3,4]. Notably, the Liberibacter lineage has also horizontally acquired a gene from the Profftella lineage, demonstrating ecological and evolutionary interactions between the HLB pathogen and the bacteriome symbiont [5]. Thus, revealing the behavior of Carsonella and Profftella during the host life cycle is essential for understanding the biology of D. citri and its associated microbiota, which would aid in the development of efficient means to control HLB.
As early as in 1937, Profft described the dynamics of apparently varied symbionts of several psyllid species using classical staining methods ( [6], reviewed in [7]). However, these methods can neither identify nor even distinguish symbiont species distinctly. Furthermore, these early papers included only a limited number of figures, all of which were hand drawn, and lacked information on D. citri. Therefore, in the present study, we analyzed the transovarial transmission and dynamics of Carsonella and Profftella during the embryonic and postembryonic development of D. citri using fluorescence in situ hybridization (FISH).

Materials and methods Psyllids
An established colony of D. citri, originally collected from Amami Oshima Island, Kagoshima, Japan, was maintained on Murraya paniculata Jack (Rutaceae) with a 16-h light period (28˚C) and 8-h dark period (23˚C). For egg collection, adult females were allowed to mate and oviposit on Citrus junos Tanaka (Rutaceae) seedlings maintained under the same conditions as described above.

Fixation and decolorization
Insect materials for FISH analysis were prepared as reported previously [8], with some modifications. Embryos, 1 st to 5 th instar nymphs, and adults were fixed in Carnoy's solution (ethanol: chloroform:glacial acetic acid, 6:3:1) at room temperature overnight. After washing with 100% ethanol, the fixed samples were treated with 6% H 2 O 2 in 80% ethanol until sufficiently decolorized. The bleached samples were then washed with 100% ethanol.

Tissue sectioning
Samples were infiltrated and embedded in polyester wax (VWR) [9], and then sliced into serial sections (5 μm thickness) using a rotary microtome RV-240 (Yamato Koki). The sections were mounted on silane-coated Platinum Pro glass slides (Matsunami Glass), and dewaxed in 100% ethanol. The samples were then rehydrated using a graded ethanol to phosphate-buffered saline (PBS) series in descending concentrations.

In situ hybridization of whole mount samples
The decolorized and washed samples were hydrated with PBSTx (0.8% NaCl, 0.02% KCl, 0.115% Na 2 HPO 4 , 0.02% KH 2 PO 4 , 0.3% Triton X-100), pre-incubated three times (20 min per incubation) with the hybridization buffer minus the probe, and then incubated with the hybridization buffer containing 100 nM of each of the probes at room temperature overnight. After washing twice with PBSTx, the samples were transferred onto glass slides with spacers, and mounted in ProLong Gold antifade reagent with DAPI using a cover slip. The specimens were examined using a Nikon A1 laser scanning confocal microscope, and acquired images were analyzed using NIS-elements AR Analysis 4.10 software (Nikon).

Results and discussion
Migration of symbionts from the bacteriome to the ovary Translocation of Carsonella and Profftella from the bacteriome to the ovary was analyzed using female adults at 0, 3, 5, and 10 days post-eclosion. In the bacteriome, as reported previously [2], Carsonella was located in uninucleate bacteriocytes [11] on the surface of the organ, while Profftella was observed in the syncytial cytoplasm, located at the center of the organ (Fig 1A  and 1B). Both Carsonella and Profftella are pleiomorphic, but are generally tubular in shape [2]. The ovaries of D. citri consist of nearly 50 ovarioles arranged in a bouquet, and are located ventrolaterally in the abdomen, just below the bacteriome [12] (Fig 1A and 1B). In adults at 0 and 3 days post-emergence, in which ovaries were small and all oocytes were at the previtellogenic stage, symbionts were observed only in the bacteriome, showing no sign of transmission to the ovary (Fig 1A).
At 5 and 10 days post-eclosion, spherical cells of both symbionts were observed in the ovary ( Fig 1B) and the hemolymph (Fig 1B and 1E, S1 Movie), in addition to the tubular Carsonella and Profftella cells in the bacteriome. These cells were presumptively in the process of transovarial transmission and migration from the bacteriome to the ovary, respectively. The ovarioles in these adults contained both previtellogenic and vitellogenic oocytes [12]. As the symbionts were observed only in the latter with accumulated yolk, it is likely that symbiont infection occurs only at the vitellogenic stage ( Fig 1B). The symbionts in the hemocoel are no longer sequestered within the host cells, enhancing their opportunity to interact with other microbes, including Liberibacter spp., which could potentially facilitate horizontal gene transfer among  [5]. In adults at 5 and 10 days post-eclosion, spherical Carsonella cells were also observed in several bacteriocytes facing the ovary, while the majority of bacteriocytes contained ordinary tubular Carsonella cells (Fig 1C, S2 Movie). This appears to reflect the transformation of Carsonella from tubular to spherical form to facilitate migration and infection into the oocyte. The mechanism of the transformation, which likely involves thickening and transverse cleavage, is yet to be elucidated, but the D. citri host must control the process as neither Carsonella nor Profftella possesses genes for cell division [2]. At the same stage (5 and 10 days post-eclosion) in the bacteriome, parts of the syncytium harboring Profftella had arrived at the surface of the organ (Fig 1D), which would facilitate the exit of Profftella from the bacteriome before migration toward the ovary (Fig 1E, S1 Movie).

Infection of oocytes by symbionts
Based on the observations described above, transmission of Carsonella and Profftella into D. citri eggs was examined using female adults at 5 days post-eclosion. Inspection of serial sections of oocytes accepting symbionts showed that a mass of Carsonella and Profftella cells entered at the posterior pole of the vitellogenic oocyte through follicle cells and the pedicel (ovariolar stalk) of the ovariole (Fig 2A-2I). This type of symbiont mass entering an oocyte is often referred to as "symbiont ball", and appears to be a common feature of transovarial transmission of various intracellular microbes, including bacteria and even yeast-like fungal symbionts, in multiple insect lineages [7,[13][14][15][16][17][18]. Interestingly, the spherical or short-rod shape of Carsonella did not change when entering the oocyte (Fig 2H, S3 Movie), whereas spherical Profftella cells transformed into a tubular form just after entering the oocyte (Fig 2I, S4 Movie). The shape of the symbionts is generally believed to reflect the duration of the intracellular symbiosis [7,19]. Buchner stated that "those (symbionts) of the syncytia, which often retain the original bacterial form, are the late-comers and that only some of them assume those bizarre growth forms as a result of long exposure to intracellular living" [7]. Carsonella is the primary symbiont, residing in all psyllid species reported to date [2,3,20,21], while Profftella, which is found only in D. citri so far, is the secondary symbiont and is therefore the late comer [2]. Nevertheless, Profftella reverted from the spherical infectious form to the "bizarre growth form" earlier than the older resident, Carsonella.

Dynamics of the symbionts during embryogenesis
Formation of the bacteriome and dynamics of the symbionts during embryogenesis were analyzed using eggs collected every 6 or 12 h from plants where adult females were allowed to mate and oviposit. In blastula-stage embryos collected 0-6 h post-oviposition, a tightly aggregated ball-like mosaic mass ("symbiont ball") of Carsonella and Profftella was observed at the posterior pole (Fig 3A). In embryos observed at 6-12 h post-oviposition, in which the newly formed germ band was at the anatrepsis stage (invagination into the central yolk) [22], the symbiont mass was loosening (Fig 3B). respectively. Carsonella cells started to elongate, and host nuclei appeared in the symbiont mass (Fig 3C and 3K). In embryos examined at 18-24 h post-oviposition, the proto-bacteriome appeared cellularized, with Carsonella and Profftella encased in the central syncytium and peripheral uninucleate bacteriocytes, respectively (Fig 3D and 3L). Notably, this arrangement is the opposite of that observed in the postembryonic bacteriome, in which Carsonella and Profftella are located in the peripheral uninuclear bacteriocytes and the central syncytium, respectively (Figs 1 and 4).
At the next stage of development (24-30 h post-oviposition), in which the head of the germ band was reaching the proto-bacteriome, uninucleate bacteriocytes harboring Profftella migrated toward the posterior pole (Fig 3E). In the subsequent katatrepsis stage (observed at 30-36 h post-oviposition), during which the embryo exits from the yolk [22], uninucleate bacteriocytes harboring Profftella assembled at the posterior pole, whereas the syncytium containing Carsonella was located on the anterior side of the mass (Fig 3F). At 36-48 h post-oviposition, embryos were in the process of dorsal closure, whereby the dorsal gap is closed by the fusion of epithelial cell sheets [22]. During this stage, the proto-bacteriome was located at the tip of the abdomen of the embryo. The syncytium, harboring Carsonella, was divided into uninuclear bacteriocytes, and was starting to surround the mass of bacteriocytes containing Profftella (Fig 3G). When the dorsal closure was completed (48-60 h post-oviposition), the proto-bacteriome was settled within the embryo, and the mass of bacteriocytes harboring Profftella was further surrounded by the uninuclear bacteriocytes containing Carsonella ( Fig  3H and 3M). When the mass of bacteriocytes harboring Profftella was completely surrounded by the Carsonella-containing bacteriocytes (60-72 h post-oviposition), the former cells were fused into a syncytium (Fig 3I and 3N). At this point, the arrangement of the symbionts was the same as that seen in the postembryonic bacteriome, with Carsonella and Profftella in the peripheral uninuclear bacteriocytes and the central syncytium, respectively. At 72-84 h postoviposition, two wing-like protrusions began to develop from both lateral sides of the bacteriome within the embryo (Fig 3J). The embryos then hatched at 84-96 h post-oviposition.
Profft studied the development of the bacteriome within Psylla alni (Psyllidae) [6], another psyllid species belonging to a different family from D. citri (Liviidae) [23]. Although few figures were presented regarding this subject, his descriptions suggest that the general trend in the formation of the bacteriome and the dynamics of symbionts, which are unidentified in P. alni, during embryogenesis is similar between the two psyllid species [6]. Whereas the mechanism of manipulating symbionts is generally unknown for insect endosymbiotic systems, the host actin was hypothesized to be involved in the process in several other previously studied insect lineages [24,25].

Postembryonic development of the bacteriome
The life cycle of D. citri includes five nymphal instars [1], each of which were examined in the current study using the whole-mount FISH method. In accordance with the increase in body size, the bacteriome continuously increased in size and volume during the nymphal  development (Fig 4A-4E). In addition, the pair of wing-like protrusions that arose in the late embryonic stage (Fig 3J) continued to grow throughout the nymphal stages (Fig 4A-4E). It appears that the shape of the bacteriome changes most dramatically during the adult eclosion, when the wing-like structures elongated further and became more eminent even than the original structure, which itself became a narrow central bridge connecting the two protrusions ( Fig 1A).