Variable organization of symbiont-containing tissue across planthoppers hosting different heritable endosymbionts.

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symbionts Sulcia and Vidania, but in many of the planthopper lineages, these symbionts are now accompanied or have been replaced by other heritable bacteria (e.g., Sodalis, Arsenophonus, Purcelliella) or fungi.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.

Alternative Transmission Patterns in Independently Acquired Nutritional Cosymbionts of Dictyopharidae Planthoppers.

Sap-sucking hemipterans host specialized, heritable microorganisms that supplement their diet with essential nutrients. These microbes show unusual features that provide a unique perspective on the coevolution of host-symbiont systems but are still poorly understood. Here, we combine microscopy with high-throughput sequencing to revisit 80-year-old reports on the diversity of symbiont transmission modes in a broadly distributed planthopper family, Dictyopharidae. We show that in seven species examined, the ancestral nutritional symbionts Sulcia and Vidania producing essential amino acids are complemented by co-primary symbionts, either Arsenophonus or Sodalis, acquired several times independently by different host lineages and contributing to the biosynthesis of B vitamins. These symbionts reside within separate bacteriomes within the abdominal cavity, although in females Vidania also occupies bacteriocytes in the rectal organ. Notably, the symbionts are transovarially transmitted from mothers to offspring in two alternative ways. In most examined species, all nutritional symbionts simultaneously infect the posterior end of the full-grown oocytes and next gather in their perivitelline space. In contrast, in other species, Sodalis colonizes the cytoplasm of the anterior pole of young oocytes, forming a cluster separate from the "symbiont ball" formed by late-invading Sulcia and Vidania. Our results show how newly arriving microbes may utilize different strategies to establish long-term heritable symbiosis. IMPORTANCE 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. We show for the first time that in a single family of planthoppers, the complementing symbionts that have established infections independently utilize different transmission strategies, one of them novel, with the transmission of different microbes separated spatially and temporally. These data show how newly arriving microbes may utilize different strategies to establish long-term heritable symbioses.

Complex symbiotic systems of two treehopper species: Centrotus cornutus (Linnaeus, 1758) and Gargara genistae (Fabricius, 1775) (Hemiptera: Cicadomorpha: Membracoidea: Membracidae).

The aim of the conducted study was to describe the symbiotic systems (the types of symbionts, distribution in the body of the host insect, the transovarial transmission between generations) of two treehoppers: Centrotus cornutus and Gargara genistae by means of microscopic and molecular techniques. We found that each of them is host to four species of bacteriome-inhabiting symbionts. In C. cornutus, ancestral bacterial symbionts Sulcia and Nasuia are accompanied by an additional symbiont-the bacterium Arsenophonus. In the bacteriomes of G. genistae, apart from Sulcia and Nasuia, bacterium Serratia is present. To our knowledge, this is the first report regarding the occurrence of Serratia as a symbiont in Hemiptera: Auchenorrhyncha. Bacteria Sulcia and Nasuia are harbored in their own bacteriocytes, whereas Arsenophonus and Serratia both inhabit their own bacteriocytes and also co-reside with bacteria Nasuia. We observed that both bacteria Arsenophonus and Serratia undergo autophagic degradation. We found that in both of the species examined, in the cytoplasm and nuclei of all of the cells of the bacteriome, bacteria Rickettsia are present. Our histological and ultrastructural observations revealed that all the bacteriome-associated symbionts of C. cornutus and G. genistae are transovarially transmitted from mother to offspring.

The influence of the essential oil extracted from hops on monolayers and bilayers imitating plant pathogen bacteria membranes.

Many plant-derived compounds possess antimicrobial, antioxidant and even anticancer activities. Therefore, they are considered as substances that can be used instead of synthetic compounds in various applications. In this work, the essential oil from hop cones was extracted and analyzed, and then its effects on model bacteria membranes were studied to verify whether the hop essential oils could be used as ecological pesticides. The experiments involved surface pressure-area measurements, penetration studies and Brewster angle microscopy (BAM) imaging of lipid monolayers as well as hydrodynamic diameter, zeta potential, steady-state fluorescence anisotropy and Cryo-Transmission Electron Microscopy (cryo-TEM) measurements of liposomes. Finally the bactericidal tests on plant pathogen bacteria Pseudomonas syringae pv. lachrymans PCM 1410 were performed. The obtained results showed that the components of the essential oils from hop cones incorporate into lipid monolayers and bilayers and alter their fluidity. However, the observed effect is determined by the system composition, its condensation and the oil concentration. Interestingly, at a given dose, the effect of the essential oil on membranes was found to stabilize. Moreover, BAM images proved that hop oil prevents the formation of a large fraction of a condensed phase at the interface. Both the studies on model membranes as well as the in vitro tests allow one to conclude that the hop essential oil could likely be considered as the candidate to be used in agriculture as a natural pesticide.

Diversity of symbiotic microbiota in Deltocephalinae leafhoppers (Insecta, Hemiptera, Cicadellidae).

Symbiotic microorganisms associated with thirteen species of the subfamily Deltocephalinae were examined using microscopic and molecular techniques. Athysanus argentarius, Euscelis incisus, Doratura stylata, Arthaldeus pascuellus, Errastunus ocellaris, Jassargus flori, Jassargus pseudocellaris, Psammotettix alienus, Psammotettix confinis, Turrutus socialis and Verdanus abdominalis harbor two types of ancient bacteriome-associated microorganisms: bacteria Sulcia (phylum Bacteroidetes) and bacteria Nasuia (phylum Proteobacteria, class Betaproteobacteria). In Balclutha calamagrostis and Balclutha punctata, the bacterium Nasuia has not been detected. In the bacteriomes of both species of Balclutha examined, only bacteria Sulcia occur, whereas Sodalis-like symbionts (phylum Proteobacteria, class Gammaproteobacteria) are localized in the fat body cells, in close vicinity of the bacteriomes. To our knowledge, this is the first report of the co-existence in Deltocephalinae leafhoppers of the ancient symbiont Sulcia and the more recently acquired Sodalis-like bacterium. The obtained results provide further evidence indicating that Deltocephalinae leafhoppers are characterized by a large diversity of symbiotic systems, which results from symbiont acquisition and replacement. The obtained results are additionally discussed in phylogenetic context.

Dual "Bacterial-Fungal" Symbiosis in Deltocephalinae Leafhoppers (Insecta, Hemiptera, Cicadomorpha: Cicadellidae).

The symbiotic systems (types of symbionts, their distribution in the host insect body, and their transovarial transmission between generations) of four Deltocephalinae leafhoppers: Fieberiella septentrionalis, Graphocraerus ventralis, Orientus ishidae, and Cicadula quadrinotata have been examined by means of histological, ultrastructural, and molecular techniques. In all four species, two types of symbionts are present: bacterium Sulcia (phylum Bacteroidetes) and yeast-like symbionts closely related to the entomopathogenic fungi (phylum Ascomycota, class Sordariomycetes). Sulcia bacteria are always harbored in giant bacteriocytes, which are grouped into large organs termed "bacteriomes." In F. septentrionalis, G. ventralis, and O. ishidae, numerous yeast-like microorganisms are localized in cells of the fat body, whereas in C. quadrinotata, they occupy the cells of midgut epithelium in large number. Additionally, in C. quadrinotata, a small amount of yeast-like microorganisms occurs intracellularly in the fat body cells and, extracellularly, in the hemolymph. Sulcia bacteria in F. septentrionalis, G. ventralis, O. ishidae, and C. quadrinotata, and the yeast-like symbionts residing in the fat body of F. septentrionalis, G. ventralis, and O. ishidae are transovarially transmitted; i.e., they infect the ovarioles which constitute the ovaries.

Sulcia symbiont of the leafhopper Macrosteles laevis (Ribaut, 1927) (Insecta, Hemiptera, Cicadellidae: Deltocephalinae) harbors Arsenophonus bacteria.

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."

Ovary of Matsucoccus pini (Insecta, Hemiptera, Coccinea: Matsucoccidae): morphology, ultrastructure, and phylogenetic implications.

The structure of ovary in a representative of the scale insect family Matsucoccidae, Matsucoccus pini, is described at the ultrastructural level. The paired ovaries of M. pini are composed of about 50 ovarioles of telotrophic type that develop asynchronously. An individual ovariole consists of an anterior tropharium (trophic chamber) and posterior vitellarium. The tropharium encloses trophocytes (nurse cells) and early previtellogenic oocytes termed arrested oocytes. In the vitellarium from 1 to 6, linearly arranged oocytes may develop. Analysis of serial sections has shown that each ovariole contains 32 germ cells (trophocytes, arrested oocytes, and developing oocytes). In the cytoplasm of all these cells, small rod-shaped bacteria are present. In the early vitellogenic oocytes, accessory nuclei arise. As vitellogenesis progresses, these nuclei migrate toward the cortical ooplasm. The obtained results are discussed in a phylogenetic context.