Anna Michalik

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.”

Bacterial symbionts of the leafhopper Evacanthus interruptus (Linnaeus, 1758) (Insecta, Hemiptera, Cicadellidae: Evacanthinae).

Plant sap-feeding hemipterans harbor obligate symbiotic microorganisms which are responsible for the synthesis of amino acids missing in their diet. In this study, we characterized the obligate symbionts hosted in the body of the xylem-feeding leafhopper Evacanthus interruptus (Cicadellidae: Evacanthinae: Evacanthini) by means of histological, ultrastructural and molecular methods. We observed that E. interruptus is associated with two types of symbiotic microorganisms: bacterium ‘Candidatus Sulcia muelleri’ (Bacteroidetes) and betaproteobacterium that is closely related to symbionts which reside in two other Cicadellidae representatives: Pagaronia tredecimpunctata (Evacanthinae: Pagaronini) and Hylaius oregonensis (Bathysmatophorinae: Bathysmatophorini). Both symbionts are harbored in their own bacteriocytes which are localized between the body wall and ovaries. In E. interruptus, both Sulcia and betaproteobacterial symbionts are transovarially transmitted from one generation to the next. In the mature female, symbionts leave the bacteriocytes and gather around the posterior pole of the terminal oocytes. Then, they gradually pass through the cytoplasm of follicular cells surrounding the posterior pole of the oocyte and enter the space between them and the oocyte. The bacteria accumulate in the deep depression of the oolemma and form a characteristic ‘symbiont ball’. In the light of the results obtained, the phylogenetic relationships within modern Cicadomorpha and some Cicadellidae subfamilies are discussed.

Bacteria belonging to the genus Burkholderia are obligatory symbionts of the eriococcids Acanthococcus aceris Signoret, 1875 and Gossyparia spuria (Modeer, 1778) (Insecta, Hemiptera, Coccoidea).

In the fat body cells of the scale insects, Gossyparia spuria and Acanthococcus aceris, numerous rod-shaped symbiotic bacteria occur. Molecular analyses have revealed that these microorganisms are closely related to the widely distributed bacterium Burkholderia. Ultrastructural observations have revealed that the bacteria are transovarially (vertically) transmitted from the mother to offspring. The microorganisms leave the fat body cells and invade ovarioles containing vitellogenic oocytes. They pass through the follicular epithelium in the neck region of the ovariole and enter the perivitelline space. Next, the symbionts infest the anterior region of the oocyte.

Ultrastructure, distribution, and transovarial transmission of symbiotic microorganisms in Nysius ericae and Nithecus jacobaeae (Heteroptera: Lygaeidae: Orsillinae)

The organization of the symbiotic system (i.e., distribution and ultrastructure of symbionts) and the mode of inheritance of symbionts in two species, Nysius ericae and Nithecus jacobaeae belonging to Heteroptera: Lygaeidae, are described. Like most hemipterans, Nysius ericae and Nithecus jacobaeae harbor obligate prokaryotic symbionts. The symbiotic bacteria are harbored in large, specialized cells termed bacteriocytes which are localized in the close vicinity of the ovaries as well as inside the ovaries. The ovaries are composed of seven ovarioles of the telotrophic type. Bacteriocytes occur in each ovariole in the basal part of tropharium termed the infection zone. The bacteriocytes form a ring surrounding the early previtellogenic oocytes. The cytoplasm of the bacteriocytes is tightly packed with large elongated bacteria. In the bacteriocytes of Nysius ericae, small, rod-shaped bacteria also occur. Both types of bacteria are transovarially transmitted from one generation to the next.

Symbiotic microorganisms in Puto superbus (Leonardi, 1907) (Insecta, Hemiptera, Coccomorpha: Putoidae)

The scale insect Puto superbus (Putoidae) lives in mutualistic symbiotic association with bacteria. Molecular phylogenetic analyses have revealed that symbionts of P. superbus belong to the gammaproteobacterial genus Sodalis. In the adult females, symbionts occur both in the bacteriocytes constituting compact bacteriomes and in individual bacteriocytes, which are dispersed among ovarioles. The bacteriocytes also house a few small, rod-shaped Wolbachia bacteria in addition to the numerous large, elongated Sodalis-allied bacteria. The symbiotic microorganisms are transovarially transmitted from generation to generation. In adult females which have choriogenic oocytes in the ovarioles, the bacteriocytes gather around the basal part of the tropharium. Next, the entire bacteriocytes pass through the follicular epithelium surrounding the neck region of the ovariole and enter the space between oocyte and follicular epithelium (perivitelline space). In the perivitelline space, the bacteriocytes assemble extracellularly in the deep depression of the oolemma at the anterior pole of the oocyte, forming a “symbiont ball”.

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.

Multiple origins of interdependent endosymbiotic complexes in a genus of cicadas

Significance Highly reduced genomes from bacteria that are long-term beneficial endosymbionts of insects often show remarkable structural stability. Endosymbionts in insects diverged by tens or hundreds of millions of years often have genomes almost completely conserved in gene order and content. Here, we show that an endosymbiont in some cicadas has repeatedly and independently fractured into complexes of distinct genomic and cellular lineages present in the same host. Individual endosymbiont lineages, having lost many of the essential ancestral genes, rely on each other for basic function and together seem to provide the same nutritional benefits as the ancestral single symbiont. These cicada endosymbionts show genomic parallels to mitochondria and provide another example of how normally stable genomes can lose structural stability. Bacterial endosymbionts that provide nutrients to hosts often have genomes that are extremely stable in structure and gene content. In contrast, the genome of the endosymbiont Hodgkinia cicadicola has fractured into multiple distinct lineages in some species of the cicada genus Tettigades. To better understand the frequency, timing, and outcomes of Hodgkinia lineage splitting throughout this cicada genus, we sampled cicadas over three field seasons in Chile and performed genomics and microscopy on representative samples. We found that a single ancestral Hodgkinia lineage has split at least six independent times in Tettigades over the last 4 million years, resulting in complexes of between two and six distinct Hodgkinia lineages per host. Individual genomes in these symbiotic complexes differ dramatically in relative abundance, genome size, organization, and gene content. Each Hodgkinia lineage retains a small set of core genes involved in genetic information processing, but the high level of gene loss experienced by all genomes suggests that extensive sharing of gene products among symbiont cells must occur. In total, Hodgkinia complexes that consist of multiple lineages encode nearly complete sets of genes present on the ancestral single lineage and presumably perform the same functions as symbionts that have not undergone splitting. However, differences in the timing of the splits, along with dissimilar gene loss patterns on the resulting genomes, have led to very different outcomes of lineage splitting in extant cicadas.

Transovarial Transmission of Symbionts in Insects

Many insects, on account of their unbalanced diet, live in obligate symbiotic associations with microorganisms (bacteria or yeast-like symbionts), which provide them with substances missing in the food they consume. In the body of host insect, symbiotic microorganisms may occur intracellularly (e.g., in specialized cells of mesodermal origin termed bacteriocytes, in fat body cells, in midgut epithelium) or extracellularly (e.g., in hemolymph, in midgut lumen). As a rule, symbionts are vertically transmitted to the next generation. In most insects, symbiotic microorganisms are transferred from mother to offspring transovarially within female germ cells. The results of numerous ultrastructural and molecular studies on symbiotic systems in different groups of insects have shown that they have a large diversity of symbiotic microorganisms and different strategies of their transmission from one generation to the next. This chapter reviews the modes of transovarial transmission of symbionts between generations in insects.

Symbiotic microorganisms of the leafhopper Deltocephalus pulicaris (Fallén, 1806) (Insecta, Hemiptera, Cicadellidae: Deltocephalinae): molecular characterization, ultrastructure and transovarial transmission*

Abstract The ovaries of the leafhopper Deltocephalus pulicaris are accompanied by large organs termed bacteriomes, which are composed of numerous polyploid cells called bacteriocytes. The cytoplasm of bacteriocytes is tightly packed with symbiotic microorganisms. Ultrastructural and molecular analyses have revealed that bacteriocytes of D. pulicaris contain two types of symbionts: the bacterium “Candidatus Sulcia muelleri” and the bacterium “Candidatus Nasuia deltocephalinicola”. Both symbionts are transovarially transmitted from the mother to the next generation.

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. Microsc. Res. Tech. 77:327–334, 2014.