The leafhopper Macrosteles laevis, like other plant sap-feeding hemipterans, lives in obligate symbiotic association with microorganisms. The symbionts are harbored in the cytoplasm of large cells termed bacteriocytes, which are integrated into huge organs termed bacteriomes. Morphological and molecular investigations have revealed that in the bacteriomes of M. laevis, two types of bacteriocytes are present which are as follows: bacteriocytes with bacterium Sulcia and bacteriocytes with Nasuia symbiont. We observed that in bacteriocytes with Sulcia, some cells of this bacterium contain numerous cells of the bacterium Arsenophonus. All types of symbionts are transmitted transovarially between generations. In the mature female, the bacteria Nasuia, bacteria Sulcia, and Sulcia with Arsenophonus inside are released from the bacteriocytes and start to assemble around the terminal oocytes. Next, the bacteria enter the cytoplasm of follicular cells surrounding the posterior pole of the oocyte. After passing through the follicular cells, the symbionts enter the space between the oocyte and follicular epithelium, forming a characteristic “symbiont ball.”

Sup-sucking hemipterans host ancient heritable microorganisms that supplement their unbalanced diet with essential nutrients and have repeatedly been complemented or replaced by other microorganisms. These symbionts need to be reliably transmitted to subsequent generations through the reproductive system, and often they end up using the same route as the most ancient ones.

Sap-feeding hemipteran insects live in associations with diverse heritable symbiotic microorganisms (bacteria and fungi) that provide essential nutrients deficient in their hosts’ diets. These symbionts typically reside in highly specialized organs called bacteriomes (with bacterial symbionts) or mycetomes (with fungal symbionts). The organization of these organs varies between insect clades that are ancestrally associated with different microbes. As these symbioses evolve and additional microorganisms complement or replace the ancient associates, the organization of the symbiont-containing tissue becomes even more variable. Planthoppers (Hemiptera: Fulgoromorpha) are ancestrally associated with bacterial symbionts Sulcia and Vidania, but in many of the planthopper lineages, these symbionts are now accompanied or have been replaced by other heritable bacteria (e.g., Sodalis, Arsenophonus, Purcelliella) or fungi. We know the identity of many of these microbes, but the symbiont distribution within the host tissues and the bacteriome organization have not been systematically studied using modern microscopy techniques. Here, we combine light, fluorescence, and transmission electron microscopy with phylogenomic data to compare symbiont tissue distributions and the bacteriome organization across planthoppers representing 15 families. We identify and describe seven primary types of symbiont localization and seven types of the organization of the bacteriome. We show that Sulcia and Vidania, when present, usually occupy distinct bacteriomes distributed within the body cavity. The more recently acquired gammaproteobacterial and fungal symbionts generally occupy separate groups of cells organized into distinct bacteriomes or mycetomes, distinct from those with Sulcia and Vidania. They can also be localized in the cytoplasm of fat body cells. Alphaproteobacterial symbionts colonize a wider range of host body habitats: Asaia-like symbionts often colonize the host gut lumen, whereas Wolbachia and Rickettsia are usually scattered across insect tissues and cell types, including cells containing other symbionts, bacteriome sheath, fat body cells, gut epithelium, as well as hemolymph. However, there are exceptions, including Gammaproteobacteria that share bacteriome with Vidania, or Alphaproteobacteria that colonize Sulcia cells. We discuss how planthopper symbiont localization correlates with their acquisition and replacement patterns and the symbionts’ likely functions. We also discuss the evolutionary consequences, constraints, and significance of these findings.

Aphidoidea, endosymbiotic bacteria, bacteriocytes, transovarial transmission of endosymbiotic bacteria The ultrastructure, distribution and transovarial transmission of endosymbiotic bacteria in representatives of six aphid families: Eriosomatidae (Pemphigus spyrothecae, Prociphilus fraxini), Anoeciidae [Anoecia (Anoecia) corni], Drepanosiphidae [Mindarus abietinus, Sipha (Rungsia) maydis, Clethrobius comes, Myzocallis (Lineomyzocallis) walshii], Thelaxidae (Thelaxes dryophila), Aphididae (Delphiniobium junackianum, Aphis viburni, Cavariella theobaldi, Macrosiphoniella tanacetaria) and Lachnidae (Schizolachnus pineti, Eulachnus rileyi) were studied at the ultrastructural level. The ovaries of aphids are accompanied by large organs termed bacteriomes that consist of giant cells termed bacteriocytes. The bacteriocyte cytoplasm is tightly packed with endosymbiotic bacteria. Ultrastructural observations have shown that the bacteria Buchnera aphidicola (primary symbiont of aphids) present in various species are characterized by significant differences in both size and organization of their cytoplasm. In the aphids, Prociphilus fraxini, Sipha (Rungsia) maydis, Thelaxes dryophila, Aphis viburni, Cavariella theobaldi, Macrosiphoniella tanacetaria, Eulachnus rileyi and Schizolachnus pineti, in addition to Buchnera aphidicola, secondary endosymbionts are also present. The bacteriocytes containing secondary endosymbionts are less numerous than those with Buchnera. In Eulachnus rileyi (Lachnidae), in addition to primary and secondary endosymbionts, there is a third type of microorganism. In all species examined both the primary and secondary endosymbionts are transovarially transmitted from mother to offspring.