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Bacteriocytes are host cells specialized to harbor symbionts in certain insect taxa. The adaptation, development, and evolution of bacteriocytes underlie insect symbiosis maintenance. Bacteriocytes carry enriched host genes of insect and bacterial origin whose transcription can be regulated by microRNAs, which are involved in host-symbiont metabolic interactions. Recognition proteins of peptidoglycan, the bacterial cell wall component, and autophagy regulate symbiont abundance in bacteriocytes. Horizontally transferred genes expressed in bacteriocytes influence the metabolism of symbiont peptidoglycan, which may affect the bacteriocyte immune response against symbionts. Bacteriocytes release or transport symbionts into ovaries for symbiont vertical transmission. Bacteriocyte development and death, regulated by transcriptional factors, are variable in different insect species. The evolutionary origin of insect bacteriocytes remains unclear. Future research should elucidate bacteriocyte cell biology, the molecular interplay between bacteriocyte metabolic and immune functions, the genetic basis of bacteriocyte origin, and the coordination between bacteriocyte function and host biology in diverse symbioses.

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The ontogenetic origins of the bacteriocytes, which are cells that harbour bacterial intracellular endosymbionts in multicellular animals, are unknown. During embryonic development, a series of morphological and transcriptional changes determine the fate of distinct cell types. The ontogeny of bacteriocytes is intimately linked with the evolutionary transition of endosymbionts from an extracellular to an intracellular environment, which in turn is linked to the diet of the host insect. Here we review the evolution and development of bacteriocytes in insects. We first classify the endosymbiotic occupants of bacteriocytes, highlighting the complex challenges they pose to the host. Then, we recall the historical account of the discovery of bacteriocytes. We then summarize the molecular interactions between the endosymbiont and the host. In addition, we illustrate the genetic contexts in which the bacteriocytes develop, with examples of the genetic changes in the hosts and endosymbionts, during specific endosymbiotic associations. We finally address the evolutionary origin as well as the putative ontogenetic or developmental source of bacteriocytes in insects.

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An important contributing factor to the evolutionary success of insects is nutritional association with microbial symbionts, which provide the host insects with nutrients lacking in their unbalanced diets. These symbionts are often compartmentalized in specialized cells of the host, the bacteriocytes. Even though bacteriocytes were first described more than a century ago, few studies have explored their dynamics throughout the insect life cycle and in response to environmental stressors. Here, we use the Buchnera aphidicola/pea aphid symbiotic system to study how bacteriocytes are regulated in response to nutritional stress throughout aphid development. Using artificial diets, we analyzed the effects of depletion or excess of phenylalanine or leucine, two amino acids essential for aphid growth and whose biosynthetic pathways are shared between the host and the symbiont. Bacteriocytes responded dynamically to those treatments, while other tissues showed no obvious morphological change. Amino acid depletion resulted in an increase in bacteriocyte numbers, with the extent of the increase depending on the amino acid, while excess either caused a decrease (for leucine) or an increase (for phenylalanine). Only a limited impact on survival and fecundity was observed, suggesting that the adjustment in bacteriocyte (and symbiont) numbers is sufficient to withstand these nutritional challenges. We also studied the impact of more extreme conditions by exposing aphids to a 24 h starvation period at the beginning of nymphal development. This led to a dramatic drop in aphid survival and fecundity and a significant developmental delay. Again, bacteriocytes responded dynamically, with a considerable decrease in number and size, correlated with a decrease in the number of symbionts, which were prematurely degraded by the lysosomal system. This study shows how bacteriocyte dynamics is integrated in the physiology of insects and highlights the high plasticity of these cells.

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Dependence on multiple nutritional symbionts that form a metabolic unit has evolved many times in insects. Although it has been postulated that host dependence on these metabolically interconnected symbionts is sustained by their high degree of anatomical integration (these symbionts are often housed in distinct symbiotic cells, the bacteriocytes, assembled into a common symbiotic organ, the bacteriome), the developmental aspects of such multipartner systems have received little attention. Aphids of the subfamilies Chaitophorinae and Lachninae typically harbor disymbiotic systems in which the metabolic capabilities of the ancient obligate symbiont Buchnera aphidicola are complemented by those of a more recently acquired nutritional symbiont, often belonging to the species Serratia symbiotica. Here, we used microscopy approaches to finely characterize the tissue tropism and infection dynamics of the disymbiotic system formed by B. aphidicola and S. symbiotica in the Norway maple aphid Periphyllus lyropictus (Chaitophorinae). Our observations show that, in this aphid, the co-obligate symbiont S. symbiotica exhibits a dual lifestyle: intracellular by being housed in large syncytial bacteriocytes embedded between B. aphidicola-containing bacteriocytes in a well-organized compartmentalization pattern, and extracellular by massively invading the digestive tract and other tissues during embryogenesis. This is the first reported case of an obligate aphid symbiont that is internalized in bacteriocytes but simultaneously adopts an extracellular lifestyle. This unusual infection pattern for an obligate insect symbiont suggests that some bacteriocyte-associated obligate symbionts, despite their integration into a cooperative partnership, still exhibit invasive behavior and escape strict compartmentalization in bacteriocytes. IMPORTANCE Multipartner nutritional endosymbioses have evolved many times in insects. In Chaitophorinae aphids, the eroded metabolic capabilities of the ancient obligate symbiont B. aphidicola are complemented by those of more recently acquired symbionts. Here, we report the atypical case of the co-obligate S. symbiotica symbiont associated with P. lyropictus. This bacterium is compartmentalized into bacteriocytes nested into the ones harboring the more ancient symbiont B. aphidicola, reflecting metabolic convergences between the two symbionts. At the same time, S. symbiotica exhibits highly invasive behavior by colonizing various host tissues, including the digestive tract during embryogenesis. The discovery of this unusual phenotype for a co-obligate symbiont reveals a new face of multipartner nutritional endosymbiosis in insects. In particular, it shows that co-obligate symbionts can retain highly invasive traits and suggests that host dependence on these bacterial partners may evolve prior to their strict compartmentalization into specialized host structures.

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Aphids are hemimetabolous insects that undergo incomplete metamorphosis without pupation. The annual life cycle of most aphids includes both an asexual (viviparous) and a sexual (oviparous) phase. Sexual reproduction only occurs once per year and is followed by many generations of asexual reproduction, during which aphids propagate exponentially with telescopic development. Here, we discuss the potential links between viviparous embryogenesis and derived developmental features in the pea aphid Acyrthosiphon pisum, particularly focusing on germline specification and axis determination, both of which are key events of early development in insects. We also discuss potential evolutionary paths through which both viviparous and oviparous females might have come to utilize maternal germ plasm to drive germline specification. This developmental strategy, as defined by germline markers, has not been reported in other hemimetabolous insects. In viviparous females, furthermore, we discuss whether molecules that in other insects characterize germ plasm, like Vasa, also participate in posterior determination and how the anterior localization of the hunchback orthologue Ap-hb establishes the anterior-posterior axis. We propose that the linked chain of developing oocytes and embryos within each ovariole and the special morphology of early embryos might have driven the formation of evolutionary novelties in germline specification and axis determination in the viviparous aphids. Moreover, based upon the finding that the endosymbiont Buchnera aphidicola is closely associated with germ cells throughout embryogenesis, we propose presumptive roles for B. aphidicola in aphid development, discussing how it might regulate germline migration in both reproductive modes of pea aphids. In summary, we expect that this review will shed light on viviparous as well as oviparous development in aphids.

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The fine structure of the female reproductive system of a cheyletid mite Bakericheyla chanayi (Trombidiformes: Cheyletidae) is investigated for the first time. This system consists of an unpaired ovary, glandular oviduct, receptaculum seminis, long cuticle-lined vagina, and genital atrium terminating in the genital opening. A separate sperm access system has not been found. The receptaculum seminis opens into the distal oviduct region, where fertilization apparently takes place. The ovary contains clusters of oogonia (cystocytes), clustered early meiotic cells, a few growing previtellogenic oocytes, and 3 large nurse cells. The dorsal ovarian region is occupied by the clusters of bacteriocytes which harbor symbiotic bacteria. Oocytes undergo vitellogenesis in individual ovarian pouches, each connected to the corresponding nurse cell by an intercellular bridge. The fine structure of the bridge suggests transport between the interconnected cells in the course of vitellogenesis. The population of cystocytes was shown to be heterogenic. The electron-light cells enter meiosis and develop into the oocytes or nurse cells. The electron-dense cystocytes do not show meiotic transformation and probably give rise to the bacteriocytes. The early development of the nurse cells and oocytes is similar and accompanied by the blebbing of the nuclear envelope, appearance of nuage material and Balbiani bodies.

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Over the past few decades, various techniques have been developed and optimized for the accurate measurement of RNA abundance in cells or tissues. These methods have been instrumental in gaining insight in complex systems such as host-symbiont associations. The pea aphid model has recently emerged as a powerful and experimentally tractable system for studying symbiotic relationships and it is the subject of a growing number of molecular studies. Nevertheless, the lack of standardized protocols for the collection of bacteriocytes, the specialized host cells harboring the symbionts, has limited its use. This chapter provides a simple, step-by-step dissection protocol for the rapid isolation of aphid bacteriocytes. We then describe an adapted protocol for efficient extraction and purification of bacteriocyte RNA that can be used for most downstream transcriptomic analyses.

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Although transovarial transmission of bacteriome-associated symbionts in hemipteran insects is extremely important for maintaining intimate host-symbiont associations, our knowledge of cellular mechanisms underlying the transmission process is quite limited. We investigated bacterial communities of salivary glands, bacteriomes, and digestive and reproductive organs and clarified the transovarial transmission of bacteriome-associated symbionts of the mountain-habitat specialist Pycna repanda using integrated methods. The bacterial communities among different gut tissues and those of bacteriomes of males and females both show similarity, whereas differences are exhibited among bacterial communities in testes and ovaries. The primary symbionts "Candidatus Sulcia muelleri" (hereafter "Ca Sulcia") and "Candidatus Hodgkinia cicadicola" (hereafter "Ca Hodgkinia") were not only restricted to but also dominant in the bacteriomes and ovaries. "Ca Hodgkinia" cells in the bacteriomes of both sexes exhibited different colors by histological and electron microscopy. Also considering the results of a restriction fragment length polymorphism (RFLP)-based cloning approach, we hypothesize that "Ca Hodgkinia" may have split into cytologically different cellular lineages within this cicada species. Regarding the dominant secondary symbionts, Rickettsia was detected in the salivary glands, digestive organs, and testes, whereas Arsenophonus was detected in the bacteriomes and ovaries. Our results show that Arsenophonus can coexist with "Ca Sulcia" and "Ca Hodgkinia" within bacteriomes and can be transovarially transmitted with these obligate symbionts together from mother to offspring in cicadas, but it is not harbored in the cytoplasm of "Ca Sulcia." The change in the shape of "Ca Sulcia" and "Ca Hodgkinia" during the transovarial transmission process is hypothesized to be related to the limited space and novel microenvironment.IMPORTANCE Cicadas establish an intimate symbiosis with microorganisms to obtain essential nutrients that are extremely deficient in host plant sap. Previous studies on bacterial communities of cicadas mainly focused on a few widely distributed species, but knowledge about mountain-habitat species is quite poor. We initially revealed the physical distribution of the primary symbionts "Ca Sulcia" and "Ca Hodgkinia" and the dominant secondary symbionts Rickettsia and Arsenophonus in the mountain-habitat specialist Pycna repanda and then clarified the transovarial transmission process of bacteriome-associated symbionts in this species. Our observations suggest that "Ca Hodgkinia" may have split into cytologically distinct lineages within this cicada species, and related cicadas might have developed complex mechanisms for the vertical transmission of the bacteriome-associated symbionts. We also revealed that Arsenophonus can be transovarially transmitted in auchenorrhynchan insects when it is not harbored in the cytoplasm of other endosymbionts. Our results highlight transovarial transmission mechanisms of bacteriome-associated symbionts in sap-feeding insects.

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Edible insects, due to their high nutritive value, are currently considered as a potential renewable source for food and feed production. Liometopum apiculatum ants are widely distributed in arid and semi-arid ecosystems and their larvae (escamoles) are considered as a delicacy, however the microbial importance in L. apiculatum nutritional ecology is unknown. The aim of this research was to characterize the microorganisms associated with both L. apiculatum larvae and the reproductive adult ants using the 16S rRNA gene sequencing and culturomics approaches. The obligate endosymbionts were also investigated through microscopic analysis. The most abundant Phylum identified by sequencing in the larvae was Firmicutes while in adult ants was Proteobacteria. Interestingly, the culturomics results showed 15 genera corresponding to the bacteria identified by sequencing analysis. Particularly, it was observed a large population of nitrogen-fixing bacteria, which could be linked with the high protein content in escamoles. Endosymbionts were detected in bacteoriocytes, these bacteria are related with vitamins and essential amino acids biosynthesis, and both compounds contributing to the high nutritional value of escamoles. This is the first report of the microorganisms present in the escamolera ant ensuring their safety as food and opening new areas of nutritional ecological and food processing.

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Symbiotic associations play a pivotal role in multicellular life by facilitating acquisition of new traits and expanding the ecological capabilities of organisms. In insects that are obligatorily dependent on intracellular bacterial symbionts, novel host cells (bacteriocytes) or organs (bacteriomes) have evolved for harboring beneficial microbial partners. The processes regulating the cellular life cycle of these endosymbiont-bearing cells, such as the cell-death mechanisms controlling their fate and elimination in response to host physiology, are fundamental questions in the biology of symbiosis. Here we report the discovery of a cell-death process involved in the degeneration of bacteriocytes in the hemipteran insect Acyrthosiphon pisum This process is activated progressively throughout aphid adulthood and exhibits morphological features distinct from known cell-death pathways. By combining electron microscopy, immunohistochemistry, and molecular analyses, we demonstrated that the initial event of bacteriocyte cell death is the cytoplasmic accumulation of nonautophagic vacuoles, followed by a sequence of cellular stress responses including the formation of autophagosomes in intervacuolar spaces, activation of reactive oxygen species, and Buchnera endosymbiont degradation by the lysosomal system. We showed that this multistep cell-death process originates from the endoplasmic reticulum, an organelle exhibiting a unique reticular network organization spread throughout the entire cytoplasm and surrounding Buchnera aphidicola endosymbionts. Our findings provide insights into the cellular and molecular processes that coordinate eukaryotic host and endosymbiont homeostasis and death in a symbiotic system and shed light on previously unknown aspects of bacteriocyte biological functioning.

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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".

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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."

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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.

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