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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.

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., Competing Interests: The authors declare no conflict of interest.