Facultatively intrabacterial localization of a planthopper endosymbiont as an adaptation to its vertical transmission.

Transovarial transmission is the most reliable way of passing on essential nutrient-providing endosymbionts from mothers to offspring. However, not all endosymbiotic microbes follow the complex path through the female host tissues to oocytes on their own. Here, we demonstrate an unusual transmission strategy adopted by one of the endosymbionts of the planthopper Trypetimorpha occidentalis (Hemiptera: Tropiduchidae) from Bulgaria. In this species, an Acetobacteraceae endosymbiont is transmitted transovarially within deep invaginations of cellular membranes of an ancient endosymbiont Sulcia-strikingly resembling recently described plant virus transmission. However, in males, Acetobacteraceae colonizes the same bacteriocytes as Sulcia but remains unenveloped. Then, the unusual endobacterial localization of Acetobacteraceae observed in females appears to be a unique adaptation to maternal transmission. Further, the symbiont's genomic features, including encoding essential amino acid biosynthetic pathways and its similarity to a recently described psyllid symbiont, suggest a unique combination of the ability to horizontally transmit among species and confer nutritional benefits. The close association with Acetobacteraceae symbiont correlates with the so-far-unreported level of genomic erosion of ancient nutritional symbionts of this planthopper. In Sulcia, this is reflected in substantial changes in genomic organization, reported for the first time in the symbiont renowned for its genomic stability. In Vidania, substantial gene loss resulted in one of the smallest genomes known, at 108.6 kb. Thus, the symbionts of T. occidentalis display a combination of unusual adaptations and genomic features that expand our understanding of how insect-microbe symbioses may transmit and evolve.IMPORTANCEReliable transmission across host generations is a major challenge for bacteria that associate with insects, and independently established symbionts have addressed this challenge in different ways. The facultatively endobacterial localization of Acetobacteraceae symbiont, enveloped by cells of ancient nutritional endosymbiont Sulcia in females but not males of the planthopper Trypetimorpha occidentalis, appears to be a unique adaptation to maternal transmission. Acetobacteraceae's genomic features indicate its unusual evolutionary history, and the genomic erosion experienced by ancient nutritional symbionts demonstrates the apparent consequences of such close association. Combined, this multi-partite symbiosis expands our understanding of the diversity of strategies that insect symbioses form and some of their evolutionary consequences.

Germline and Somatic Cell Syncytia in Insects.

Syncytia are common in the animal and plant kingdoms both under normal and pathological conditions. They form through cell fusion or division of a founder cell without cytokinesis. A particular type of syncytia occurs in invertebrate and vertebrate gametogenesis when the founder cell divides several times with partial cytokinesis producing a cyst (nest) of germ line cells connected by cytoplasmic bridges. The ultimate destiny of the cyst's cells differs between animal groups. Either all cells of the cyst become the gametes or some cells endoreplicate or polyploidize to become the nurse cells (trophocytes). Although many types of syncytia are permanent, the germ cell syncytium is temporary, and eventually, it separates into individual gametes. In this chapter, we give an overview of syncytium types and focus on the germline and somatic cell syncytia in various groups of insects. We also describe the multinuclear giant cells, which form through repetitive nuclear divisions and cytoplasm hypertrophy, but without cell fusion, and the accessory nuclei, which bud off the oocyte nucleus, migrate to its cortex and become included in the early embryonic syncytium.

Nutrient supplementation by genome-eroded Burkholderia symbionts of scale insects.

Hemipterans are known as hosts to bacterial or fungal symbionts that supplement their unbalanced diet with essential nutrients. Among them, scale insects (Coccomorpha) are characterized by a particularly large diversity of symbiotic systems. Here, using microscopic and genomic approaches, we functionally characterized the symbionts of two scale insects belonging to the Eriococcidae family, Acanthococcus aceris and Gossyparia spuria. These species host Burkholderia bacteria that are localized in the cytoplasm of the fat body cells. Metagenome sequencing revealed very similar and highly reduced genomes (<900KBp) with a low GC content (~38%), making them the smallest and most AT-biased Burkholderia genomes yet sequenced. In their eroded genomes, both symbionts retain biosynthetic pathways for the essential amino acids leucine, isoleucine, valine, threonine, lysine, arginine, histidine, phenylalanine, and precursors for the semi-essential amino acid tyrosine, as well as the cobalamin-dependent methionine synthase MetH. A tryptophan biosynthesis pathway is conserved in the symbiont of G. spuria, but appeared pseudogenized in A. aceris, suggesting differential availability of tryptophan in the two host species' diets. In addition to the pathways for essential amino acid biosynthesis, both symbionts maintain biosynthetic pathways for multiple cofactors, including riboflavin, cobalamin, thiamine, and folate. The localization of Burkholderia symbionts and their genome traits indicate that the symbiosis between Burkholderia and eriococcids is younger than other hemipteran symbioses, but is functionally convergent. Our results add to the emerging picture of dynamic symbiont replacements in sap-sucking Hemiptera and highlight Burkholderia as widespread and versatile intra- and extracellular symbionts of animals, plants, and fungi.

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.

Ovary structure and symbiotic associates of a ground mealybug, Rhizoecus albidus (Hemiptera, Coccomorpha: Rhizoecidae) and their phylogenetic implications.

The ovary structure and the organization of its symbiotic system of the ground mealybug, Rhizoecus albidus (Rhizoecidae), were examined by means of microscopic and molecular methods. Each of the paired elongated ovaries of R. albidus is composed of circa one hundred short telotrophic-meroistic ovarioles, which are radially arranged along the distal part of the lateral oviduct. Analysis of serial sections revealed that each ovariole contains four germ cells: three trophocytes (nurse cells) occupying the tropharium and a single oocyte in the vitellarium. The ovaries are accompanied by giant cells termed bacteriocytes which are tightly packed with large pleomorphic bacteria. Their identity as Brownia rhizoecola (Bacteroidetes) was confirmed by means of amplicon sequencing and fluorescence in situ hybridization techniques. Moreover, to our knowledge, this is the first report on the morphology and ultrastructure of the Brownia rhizoecola bacterium. In the bacteriocyte cytoplasm bacteria Brownia co-reside with sporadic rod-shaped smaller bacteria, namely Wolbachia (Proteobacteria: Alphaproteobacteria). Both symbionts are transmitted to the next generation vertically (maternally), that is, via female germline cells. We documented that, at the time when ovarioles contain oocytes at the vitellogenic stage, these symbionts leave the bacteriocytes and move toward the neck region of ovarioles (i.e. the region between tropharium and vitellarium). Next, the bacteria enter the cytoplasm of follicular cells surrounding the basal part of the tropharium, leave them and enter the space between the follicular epithelium and surface of the nutritive cord connecting the tropharium and vitellarium. Finally, they gather in the deep depression of the oolemma at the anterior pole of the oocyte in the form of a 'symbiont ball'. Our results provide further arguments strongly supporting the validity of the recent changes in the classification of mealybugs, which involved excluding ground mealybugs from the Pseudococcidae family and raising them to the rank of their own family Rhizoecidae.

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.

Fungal Associates of Soft Scale Insects (Coccomorpha: Coccidae).

Ophiocordyceps fungi are commonly known as virulent, specialized entomopathogens; however, recent studies indicate that fungi belonging to the Ophiocordycypitaceae family may also reside in symbiotic interaction with their host insect. In this paper, we demonstrate that Ophiocordyceps fungi may be obligatory symbionts of sap-sucking hemipterans. We investigated the symbiotic systems of eight Polish species of scale insects of Coccidae family: Parthenolecanium corni, Parthenolecanium fletcheri, Parthenolecanium pomeranicum, Psilococcus ruber, Sphaerolecanium prunasti, Eriopeltis festucae, Lecanopsis formicarum and Eulecanium tiliae. Our histological, ultrastructural and molecular analyses showed that all these species host fungal symbionts in the fat body cells. Analyses of ITS2 and Beta-tubulin gene sequences, as well as fluorescence in situ hybridization, confirmed that they should all be classified to the genus Ophiocordyceps. The essential role of the fungal symbionts observed in the biology of the soft scale insects examined was confirmed by their transovarial transmission between generations. In this paper, the consecutive stages of fungal symbiont transmission were analyzed under TEM for the first time.

The Diversity of Symbiotic Systems in Scale Insects.

Most scale insects, like many other plant sap-sucking hemipterans, harbor obligate symbionts of bacterial or fungal origin, which synthesize and provide the host with substances missing in their restricted diet. Histological, ultrastructural, and molecular analyses have revealed that scale insects differ in the type of symbionts, the localization of symbionts in the host body, and the mode of transmission of symbionts from one generation to the next. Symbiotic microorganisms may be distributed in the cells of the fat body, midgut epithelium, inside the cells of other symbionts, or the specialized cells of a mesodermal origin, termed bacteriocytes. In most scale insects, their symbiotic associates are inherited transovarially, wherein the mode of transmission may have a different course-the symbionts may invade larval ovaries containing undifferentiated germ cells or ovaries of adult females containing vitellogenic or choriogenic oocytes.

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.

Molecular characterization, ultrastructure, and transovarial transmission of Tremblaya phenacola in six mealybugs of the Phenacoccinae subfamily (Insecta, Hemiptera, Coccomorpha).

Mealybugs (Hemiptera, Coccomorpha: Pseudococcidae) are plant sap-sucking insects which require close association with nutritional microorganisms for their proper development and reproduction. Here, we present the results of histological, ultrastructural, and molecular analyses of symbiotic systems of six mealybugs belonging to the Phenacoccinae subfamily: Phenacoccus aceris, Rhodania porifera, Coccura comari, Mirococcus clarus, Peliococcus calluneti, and Ceroputo pilosellae. Molecular analyses based on bacterial 16S rRNA genes have revealed that all the investigated species of Phenacoccinae are host to only one type of symbiotic bacteria-a large pleomorphic betaproteobacteria-Tremblaya phenacola. In all the species examined, bacteria are localized in the specialized cells of the host-insect termed bacteriocytes and are transovarially transmitted between generations. The mode of transovarial transmission is similar in all of the species investigated. Infection takes place in the neck region of the ovariole, between the tropharium and vitellarium. The co-phylogeny between mealybugs and bacteria Tremblaya has been also analyzed.

Bacterial associates of Orthezia urticae, Matsucoccus pini, and Steingelia gorodetskia - scale insects of archaeoccoid families Ortheziidae, Matsucoccidae, and Steingeliidae (Hemiptera, Coccomorpha).

The biological nature, ultrastructure, distribution, and mode of transmission between generations of the microorganisms associated with three species (Orthezia urticae, Matsucoccus pini, Steingelia gorodetskia) of primitive families (archaeococcoids = Orthezioidea) of scale insects were investigated by means of microscopic and molecular methods. In all the specimens of Orthezia urticae and Matsucoccus pini examined, bacteria Wolbachia were identified. In some examined specimens of O. urticae, apart from Wolbachia, bacteria Sodalis were detected. In Steingelia gorodetskia, the bacteria of the genus Sphingomonas were found. In contrast to most plant sap-sucking hemipterans, the bacterial associates of O. urticae, M. pini, and S. gorodetskia are not harbored in specialized bacteriocytes, but are dispersed in the cells of different organs. Ultrastructural observations have shown that bacteria Wolbachia in O. urticae and M. pini, Sodalis in O. urticae, and Sphingomonas in S. gorodetskia are transovarially transmitted from mother to progeny.

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.

Symbiotic cornucopia of the monophagous planthopper Ommatidiotus dissimilis (Fallén, 1806) (Hemiptera: Fulgoromorpha: Caliscelidae).

In contrast to Cicadomorpha, in which numerous symbiotic bacteria have been identified and characterized, the symbionts of fulgoromorphans are poorly known. Here, we present the results of histological, ultrastructural, and molecular analyses of the symbiotic system of the planthopper Ommatidiotus dissimilis. Amplification, cloning, and sequencing of bacterial 16S RNA genes have revealed that O. dissimilis is host to five types of bacteria. Apart from bacteria Sulcia and Vidania, which are regarded as ancestral symbionts of Fulgoromorpha, three additional types of bacteria belonging to the genera Sodalis, Wolbachia, and Rickettsia have been detected. Histological and ultrastructural investigations have shown that bacteria Sulcia, Vidania, and Sodalis house separate bacteriocytes, whereas bacteria Wolbachia and Rickettsia are dispersed within various insect tissue. Additionally, bacteria belonging to the genus Vidania occupy the bacteriome localized in the lumen of the hindgut. Both molecular and microscopic analyses have revealed that all the symbionts are transovarially transmitted between generations.

Coexistence of novel gammaproteobacterial and Arsenophonus symbionts in the scale insect Greenisca brachypodii (Hemiptera, Coccomorpha: Eriococcidae).

Scale insects are commonly associated with obligate, intracellular microorganisms which play important roles in complementing their hosts with essential nutrients. Here we characterized the symbiotic system of Greenisca brachypodii, a member of the family Eriococcidae. Histological and ultrastructural analyses have indicated that G. brachypodii is stably associated with coccoid and rod-shaped bacteria. Phylogenetic analyses have revealed that the coccoid bacteria represent a sister group to the secondary symbiont of the mealybug Melanococcus albizziae, whereas the rod-shaped symbionts are close relatives of Arsenophonus symbionts in insects - to our knowledge, this is the first report of the presence of Arsenophonus bacterium in scale insects. As a comparison of 16S and 23S rRNA genes sequences of the G. brachypodii coccoid symbiont with other gammaprotebacterial sequences showed only low similarity (∼90%), we propose the name 'Candidatus Kotejella greeniscae' for its tentative classification. Both symbionts are transovarially transmitted from one generation to the next. The infection takes place in the neck region of the ovariole. The bacteria migrate between follicular cells, as well as through the cytoplasm of those cells to the perivitelline space, where they form a characteristic 'symbiont ball'. Our findings provide evidence for a polyphyletic origin of symbionts of Eriococcidae.

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.

Yeast-like microorganisms in the scale insect Kermes quercus (Insecta, Hemiptera, Coccomorpha: Kermesidae). Newly acquired symbionts?

Scale insects, like other plant sap-consumers, are host to symbiotic microorganisms which provide them with the substances missing from their diet. In contrast to most scale insects, Kermes quercus (Linnaeus) was regarded as asymbiotic. Our histological and ultrastructural observations show that in the body of the feeding stages of K. quercus collected in two locations (Warsaw and Cracow), numerous yeast-like microorganisms occur. These microorganisms were localized in the cytoplasm of fat body cells. The yeast-like microorganisms were observed neither in other organs of the host insect nor in the eggs. These microorganisms did not cause any damage to the structure of the ovaries and the course of oogenesis of the host insect. The females infected by them produced about 1300 larvae. The lack of these microorganisms in the cytoplasm of eggs indicates that they are not transmitted transovarially from mother to offspring. Molecular analyses indicated that the microorganisms which reside in the body of K. quercus are closely related to the entomopathogenic fungi Cordyceps and Ophiocordyceps, which belong to the Sordariomycetes class within the Ascomycota. The role of yeast-like microorganisms to their host insects remains unknown; however, it has been suggested that they may represent newly acquired symbionts.

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.

Development of ovary structures in the last larval and adult stages of psyllids (Insecta, Hemiptera, Sternorrhyncha: Psylloidea).

The development and organization of the ovaries of ten species from four Psylloidea families (Psyllidae, Triozidae, Aphalaridae and Liviidae) have been investigated. The ovaries of the last larval stage (i.e. fifth instar) of all examined species are filled with numerous clusters of cystocytes which undergo synchronous incomplete mitotic division. Cystocytes of the given cluster are arranged into a rosette with polyfusome in the centre. These clusters are associated with single somatic cells. At the end of the fifth instar, the clusters begin to separate from each other, forming spherical ovarioles which are surrounded by a single layer of somatic cells. In the ovarioles of very young females all cystocytes enter the prophase of meiosis and differentiate shortly thereafter into oocytes and trophocytes (nurse cells). Meanwhile, somatic cells differentiate into cells of the inner epithelial sheath surrounding the trophocytes and into the prefollicular cells that encompass the oocytes. During this final differentiation, the trophocytes lose their cell membranes and become syncytial. Oocytes remain cellular and most of them (termed arrested oocytes) do not grow. In the ovarioles of older females, one oocyte encompassed by its follicle cells starts growing, still connected to the syncytial tropharium by a nutritive cord. After the short phase of previtellogenesis alone, the oocyte enters its vitellogenic the growth phase in the vitellarium. At that time, the second oocyte may enter the vitellarium and start its previtellogenic growth. In the light of the obtained results, the phylogeny of psyllids, as well as phylogenetic relationships between taxa of Hemiptera: Sternorrhyncha 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.