Host-Encoded Aminotransferase Import into the Endosymbiotic Bacteria Nardonella of Red Palm Weevil.

Symbiotic systems are intimately integrated at multiple levels. Host-endosymbiont metabolic complementarity in amino acid biosynthesis is especially important for sap-feeding insects and their symbionts. In weevil-Nardonella endosymbiosis, the final step reaction of the endosymbiont tyrosine synthesis pathway is complemented by host-encoded aminotransferases. Based on previous results from other insects, we suspected that these aminotransferases were likely transported into the Nardonella cytoplasm to produce tyrosine. Here, we identified five aminotransferase genes in the genome of the red palm weevil. Using quantitative real-time RT-PCR, we confirmed that RfGOT1 and RfGOT2A were specifically expressed in the bacteriome. RNA interference targeting these two aminotransferase genes reduced the tyrosine level in the bacteriome. The immunofluorescence-FISH double labeling localization analysis revealed that RfGOT1 and RfGOT2A were present within the bacteriocyte, where they colocalized with Nardonella cells. Immunogold transmission electron microscopy demonstrated the localization of RfGOT1 and RfGOT2A in the cytosol of Nardonella and the bacteriocyte. Our data revealed that RfGOT1 and RfGOT2A are transported into the Nardonella cytoplasm to collaborate with genes retained in the Nardonella genome in order to synthesize tyrosine. The results of our study will enhance the understanding of the integration of host and endosymbiont metabolism in amino acid biosynthesis.

A bacteriocyte symbiont determines whitefly sex ratio by regulating mitochondrial function.

Nutritional symbionts influence host reproduction, but the underlying molecular mechanisms are largely unclear. We previously found that the bacteriocyte symbiont Hamiltonella impacts the sex ratio of the whitefly Bemisia tabaci. Hamiltonella synthesizes folate by cooperation with the whitefly. Folate deficiency by Hamiltonella elimination or whitefly gene silencing distorted whitefly sex ratio, and folate supplementation restored the sex ratio. Hamiltonella deficiency or gene silencing altered histone H3 lysine 9 trimethylation (H3K9me3) level, which was restored by folate supplementation. Genome-wide chromatin immunoprecipitation-seq analysis of H3K9me3 indicated mitochondrial dysfunction in symbiont-deficient whiteflies. Hamiltonella deficiency compromised mitochondrial quality of whitefly ovaries. Repressing ovary mitochondrial function led to distorted whitefly sex ratio. These findings indicate that the symbiont-derived folate regulates host histone methylation modifications, which thereby impacts ovary mitochondrial function, and finally determines host sex ratio. Our study suggests that a nutritional symbiont can regulate animal reproduction in a way that differs from reproductive manipulators.

Vitellogenin Facilitates Associations between the Whitefly and a Bacteriocyte Symbiont.

Integration between animal reproduction and symbiont inheritance is fundamental in symbiosis biology, but the underlying molecular mechanisms are largely unknown. Vitellogenin (Vg) is critical for oogenesis, and it is also a pathogen pattern recognition molecule in some animals. Previous studies have shown that Vg is involved in the regulation of symbiont abundance and transmission. However, the mechanisms by which an insect and its symbiont contribute to the function of Vg and how Vg impacts the persistence of insect-microbe symbiosis remain largely unclear. Symbionts are transovarially transmitted via maternal inheritance of the bacteriocytes in the whitefly Bemisia tabaci. Surprisingly, Vg is localized in bacteriocytes of whiteflies. Vg could be synthesized in whitefly bacteriocytes by the gene Vg expressed in these cells or exported into bacteriocytes from hemolymph via the Vg receptor. We further found that the juvenile hormone and "Candidatus Portiera aleyrodidarum" (here termed Portiera) control the level and localization of Vg in whiteflies. Immunocapture PCR revealed interactions between Vg and Portiera. Suppressing Vg expression reduced Portiera abundance as well as whitefly oogenesis and fecundity. Thus, we reveal that Vg facilitated the persistence of whitefly-bacteriocyte symbiont associations. This study will provide insight into the key role of Vg in the coevolution of insect reproduction and symbiont inheritance. IMPORTANCE Intracellular heritable symbionts have been incorporated into insect reproductive and developmental biology by various mechanisms. All Bemisia tabaci species harbor the obligate symbiont Portiera in specialized insect cells called bacteriocytes. We report that the whitefly juvenile hormone and Portiera determined vitellogenin (Vg) localization in bacteriocytes of whiteflies. In turn, Vg affected whitefly fecundity as well as fitness and transmission of the symbiont. Our findings show that Vg, a multifunctional protein, is indispensable for symbiont integration into the reproduction and development of insects. This reflects the outcome of long-term coevolution of the insect-microbe symbiosis.

Bacteriocyte development is sexually differentiated in Bemesia tabaci.

Some symbiotic microbes are restricted to specialized host cells called bacteriocytes. However, the molecular and cellular mechanisms underlying the development of bacteriocytes are largely obscure. We find that maternally inherited bacteriocytes proliferate in adult females but degenerate in adult males of the whitefly Bemisia tabaci. Single-cell transcriptomics and immunohistochemistry reveal that cell division only occurs in the bacteriocytes of adult females, whereas autophagy and apoptosis are induced in the bacteriocytes of adult males. A transcription factor, Adf-1, enriched in bacteriocytes, is highly expressed in female bacteriocytes relative to male bacteriocytes. Silencing Adf-1 reduces the bacteriocyte number and Portiera titer and activates autophagy and apoptosis in females. The differential dynamics of both cell division and death in bacteriocytes and distinct expression of Adf-1 in bacteriocytes between whitefly sexes underlie the sexual differentiation of bacteriocyte development. Our study reveals that insect sex affects the development of bacteriocytes by cellular and molecular remodeling.

A novel microRNA regulates cooperation between symbiont and a laterally acquired gene in the regulation of pantothenate biosynthesis within Bemisia tabaci whiteflies.

Horizontally transferred genes (HTGs) play a key role in animal symbiosis, and some horizontally transferred genes or proteins are highly expressed in specialized host cells (bacteriocytes). However, it is not clear how HTGs are regulated, but microRNAs (miRNAs) are prime candidates given their previously demonstrated roles in symbiosis and impacts on the expression of host genes. A horizontally acquired PanBC that is highly expressed in whitefly bacteriocytes can cooperate with an obligate symbiont Portiera for pantothenate production, facilitating whitefly performance and Portiera titre. Here, we found that a whitefly miRNA, novel-m0780-5p, was up-regulated and its target panBC was down-regulated in Portiera-eliminated whiteflies. This miRNA was located in the cytoplasmic region of whitefly bacteriocytes. Injection of novel-m0780-5p agomir reduced the expression of PanBC in whitefly bacteriocytes, while injection of novel-m0780-5p antagomir enhanced PanBC expression. Agomir injection also reduced the pantothenate level, Portiera titre and whitefly performance. Supplementation with pantothenate restored Portiera titre and the fitness of agomir-injected whiteflies. Thus, we demonstrate that a whitefly miRNA regulates panBC-mediated host-symbiont collaboration required for pantothenate synthesis, benefiting the whitefly-Portiera symbiosis. Both panBC and novel-m0780-5p are present in the genomes of six Bemisia tabaci species. The expression of a novel miRNA in multiple B. tabaci species suggests that the miRNA evolved after panBC acquisition, and allowed this gene to be more tightly regulated. Our discovery provides the first account of a HTG being regulated by a miRNA from the host genome, and suggests key roles for interactions between miRNAs and HTGs in the functioning of symbiosis.

Autophagy Regulates Whitefly-Symbiont Metabolic Interactions.

Nutritional symbionts are restricted to specialized host cells called bacteriocytes in various insect orders. These symbionts can provide essential nutrients to the host. However, the cellular mechanisms underlying the regulation of these insect-symbiont metabolic associations remain largely unclear. The whitefly Bemisia tabaci MEAM1 hosts "Candidatus Portiera aleyrodidarum" (here, "Ca. Portiera") and "Candidatus Hamiltonella defensa" (here, "Ca. Hamiltonella") bacteria in the same bacteriocyte. In this study, the induction of autophagy by chemical treatment and gene silencing decreased symbiont titers and essential amino acid (EAA) and B vitamin contents. In contrast, the repression of autophagy in bacteriocytes via Atg8 silencing increased symbiont titers, and amino acid and B vitamin contents. Furthermore, dietary supplementation with non-EAAs or B vitamins alleviated autophagy in whitefly bacteriocytes, elevated TOR (target of rapamycin) expression, and increased symbiont titers. TOR silencing restored symbiont titers in whiteflies after dietary supplementation with B vitamins. These data suggest that "Ca. Portiera" and "Ca. Hamiltonella" evade autophagy of the whitefly bacteriocytes by activating the TOR pathway via providing essential nutrients. Taken together, we demonstrate that autophagy plays a critical role in regulating the metabolic interactions between the whitefly and two intracellular symbionts. Therefore, this study reveals that autophagy is an important cellular basis for bacteriocyte evolution and symbiosis persistence in whiteflies. The whitefly symbiosis unravels the interactions between cellular and metabolic functions of bacteriocytes. IMPORTANCE Nutritional symbionts, which are restricted to specialized host cells called bacteriocytes, can provide essential nutrients for many hosts. However, the cellular mechanisms of regulation of animal-symbiont metabolic associations have been largely unexplored. Here, using the whitefly-"Ca. Portiera"/"Ca. Hamiltonella" endosymbiosis, we demonstrate autophagy regulates the symbiont titers and thereby alters the essential amino acid and B vitamin contents. For persistence in the whitefly bacteriocytes, "Ca. Portiera" and "Ca. Hamiltonella" alleviate autophagy by activating the TOR (target of rapamycin) pathway through providing essential nutrients. Therefore, we demonstrate that autophagy plays a critical role in regulating the metabolic interactions between the whitefly and two intracellular symbionts. This study also provides insight into the cellular basis of bacteriocyte evolution and symbiosis persistence in the whitefly. The mechanisms underlying the role of autophagy in whitefly symbiosis could be widespread in many insect nutritional symbioses. These findings provide a new avenue for whitefly control via regulating autophagy in the future.

Vertical Transmission at the Pathogen-Symbiont Interface: Serratia symbiotica and Aphids.

Many insects possess beneficial bacterial symbionts that occupy specialized host cells and are maternally transmitted. As a consequence of their host-restricted lifestyle, these symbionts often possess reduced genomes and cannot be cultured outside hosts, limiting their study. The bacterial species Serratia symbiotica was originally characterized as noncultured strains that live as mutualistic symbionts of aphids and are vertically transmitted through transovarial endocytosis within the mother's body. More recently, culturable strains of S. symbiotica were discovered that retain a larger set of ancestral Serratia genes, are gut pathogens in aphid hosts, and are principally transmitted via a fecal-oral route. We find that these culturable strains, when injected into pea aphids, replicate in the hemolymph and are pathogenic. Unexpectedly, they are also capable of maternal transmission via transovarial endocytosis: using green fluorescent protein (GFP)-tagged strains, we observe that pathogenic S. symbiotica strains, but not Escherichia coli, are endocytosed into early embryos. Furthermore, pathogenic S. symbiotica strains are compartmentalized into specialized aphid cells in a fashion similar to that of mutualistic S. symbiotica strains during later stages of embryonic development. However, infected embryos do not appear to develop properly, and offspring infected by a transovarial route are not observed. Thus, cultured pathogenic strains of S. symbiotica have the latent capacity to transition to lifestyles as mutualistic symbionts of aphid hosts, but persistent vertical transmission is blocked by their pathogenicity. To transition into stably inherited symbionts, culturable S. symbiotica strains may need to adapt to regulate their titer, limit their pathogenicity, and/or provide benefits to aphids that outweigh their cost.IMPORTANCE Insects have evolved various mechanisms to reliably transmit their beneficial bacterial symbionts to the next generation. Sap-sucking insects, including aphids, transmit symbionts by endocytosis of the symbiont into cells of the early embryo within the mother's body. Experimental studies of this process are hampered by the inability to culture or genetically manipulate host-restricted, symbiotic bacteria. Serratia symbiotica is a bacterial species that includes strains ranging from obligate, heritable symbionts to gut pathogens. We demonstrate that culturable S. symbiotica strains, which are aphid gut pathogens, can be maternally transmitted. Cultured S. symbiotica therefore possesses a latent capacity for evolving a host-restricted lifestyle and can be used to understand the transition from pathogenicity to beneficial symbiosis.

Bacteriome-Associated Endosymbiotic Bacteria of Nosodendron Tree Sap Beetles (Coleoptera: Nosodendridae).

The family Nosodendridae is a small group of tree sap beetles with only 91 described species representing three genera from the world. In 1930s, bacteria-harboring symbiotic organs, called bacteriomes, were briefly described in a European species Nosodendron fasciculare. Since then, however, no studies have been conducted on the nosodendrid endosymbiosis for decades. Here we investigated the bacteriomes and the endosymbiotic bacteria of Nosodendron coenosum and Nosodendron asiaticum using molecular phylogenetic and histological approaches. In adults and larvae, a pair of slender bacteriomes were found along both sides of the midgut. The bacteriomes consisted of large bacteriocytes at the center and flat sheath cells on the surface. Fluorescence in situ hybridization detected preferential localization of the endosymbiotic bacteria in the cytoplasm of the bacteriocytes. In reproductive adult females, the endosymbiotic bacteria were also detected at the infection zone in the ovarioles and on the surface of growing oocytes, indicating vertical symbiont transmission via ovarial passage. Transmission electron microscopy unveiled bizarre structural features of the bacteriocytes, whose cytoplasm exhibited degenerate cytology with deformed endosymbiont cells. Molecular phylogenetic analysis revealed that the nosodendrid endosymbionts formed a distinct clade in the Bacteroidetes. The nosodendrid endosymbionts were the most closely related to the bacteriome endosymbionts of bostrichid powderpost beetles and also allied to the bacteriome endosymbionts of silvanid grain beetles, uncovering an unexpected endosymbiont relationship across the unrelated beetle families Nosodendridae, Bostrichidae and Silvanidae. Host-symbiont co-evolution and presumable biological roles of the endosymbiotic bacteria are discussed.

Bacteriocytes and Blattabacterium Endosymbionts of the German Cockroach Blattella germanica, the Forest Cockroach Blattella nipponica, and Other Cockroach Species.

Cockroaches are commonly found in human residences and notorious as hygienic and nuisance pests. Notably, however, no more than 30 cockroach species are regarded as pests, while the majority of 4,500 cockroaches in the world are living in forest environments with little relevance to human life. Why some cockroaches have exceptionally adapted to anthropic environments and established pest status is of interest. Here we investigated the German cockroach Blattella germanica, which is a cosmopolitan pest species, and the forest cockroach Blattella nipponica, which is a wild species closely related to B. germanica. In contrast to easy rearing of B. germanica, laboratory rearing of B. nipponica was challenging-several trials enabled us to keep the insects for up to three months. We particularly focused on the distribution patterns of specialized cells, bacteriocytes, for harboring endosymbiotic Blattabacterium, which has been suggested to contribute to host's nitrogen metabolism and recycling, during the postembryonic development of the insects. The bacteriocytes were consistently localized to visceral fat bodies filling the abdominal body cavity, where a number of single bacteriocytes were scattered among the adipocytes, throughout the developmental stages in both females and males. The distribution patterns of the bacteriocytes were quite similar between B. germanica and B. nipponica, and also among other diverse cockroach species, plausibly reflecting the highly conserved cockroach-Blattabacterium symbiotic association over evolutionary time. Our study lays a foundation to experimentally investigate the origin and the processes of urban pest evolution, on account of possible involvement of microbial associates.

Cellular Localization of Two Rickettsia Symbionts in the Digestive System and within the Ovaries of the Mirid Bug, Macrolophous pygmaeus.

Bacterial symbionts in arthropods are common, vary in their effects, and can dramatically influence the outcome of biological control efforts. Macrolophus pygmaeus (Heteroptera: Miridae), a key component of biological control programs, is mainly predaceous but may also display phytophagy. M. pygmaeus hosts symbiotic Wolbachia, which induce cytoplasmic incompatibility, and two Rickettsia species, R. bellii and R. limoniae, which are found in all individuals tested. To test possible involvement of the two Rickettsia species in the feeding habits of M. pygmaeus, we first showed that the microbiome of the insect is dominated by these three symbionts, and later described the distribution pattern of the two Rickettsia species in its digestive system. Although both Rickettsia species were located in certain gut bacteriocyes, in caeca and in Malpighian tubules of both sexes, each species has a unique cellular occupancy pattern and specific distribution along digestive system compartments. Infrequently, both species were found in a cell. In females, both Rickettsia species were detected in the germarium, the apical end of the ovarioles within the ovaries, but not in oocytes. Although the cause for these Rickettsia distribution patterns is yet unknown, it is likely linked to host nutrition while feeding on prey or plants.

Patterns of host cell inheritance in the bacterial symbiosis of whiteflies.

Whiteflies possess bacterial symbionts Candidatus Portiera aleyrodidium that are housed in specialized cells called bacteriocytes and are faithfully transmitted via the ovary to insect offspring. In one whitefly species studied previously, Bemisia tabaci MEAM1, transmission is mediated by somatic inheritance of bacteriocytes, with a single bacteriocyte transferred to each oocyte and persisting through embryogenesis to the next generation. Here, we investigate the mode of bacteriocyte transmission in two whitefly species, B. tabaci MED, the sister species of MEAM1, and the phylogenetically distant species Trialeurodes vaporariorum. Microsatellite analysis supported by microscopical studies demonstrates that B. tabaci MED bacteriocytes are genetically different from other somatic cells and persist through embryogenesis, as for MEAM1, but T. vaporariorum bacteriocytes are genetically identical to other somatic cells of the insect, likely mediated by the degradation of maternal bacteriocytes in the embryo. These two alternative modes of transmission provide a first demonstration among insect symbioses that the cellular processes underlying vertical transmission of bacterial symbionts can diversify among related host species associated with a single lineage of symbiotic bacteria.

microRNA-92a regulates the expression of aphid bacteriocyte-specific secreted protein 1.

OBJECTIVE: Aphids harbor a nutritional obligate endosymbiont in specialized cells called bacteriocytes, which aggregate to form an organ known as the bacteriome. Aphid bacteriomes display distinct gene expression profiles that facilitate the symbiotic relationship. Currently, the mechanisms that regulate these patterns of gene expression are unknown. Recently using computational pipelines, we identified miRNAs that are conserved in expression in the bacteriomes of two aphid species and proposed that they function as important regulators of bacteriocyte gene expression. Here using a dual luciferase assay in mouse NIH/3T3 cell culture, we aimed to experimentally validate the computationally predicted interaction between Myzus persicae miR-92a and the predicted target region of M. persicae bacteriocyte-specific secreted protein 1 (SP1) mRNA. RESULTS: In the dual luciferase assay, miR-92a interacted with the SP1 target region resulting in a significant downregulation of the luciferase signal. Our results demonstrate that miR-92a interacts with SP1 to alter expression in a heterologous expression system, thereby supporting our earlier assertion that miRNAs are regulators of the aphid/Buchnera symbiotic interaction.

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.

Antimicrobial Activities of Cysteine-rich Peptides Specific to Bacteriocytes of the Pea Aphid Acyrthosiphon pisum.

Aphids have a mutualistic relationship with the bacterial endosymbiont Buchnera aphidicola. We previously reported seven cysteine-rich peptides in the pea aphid Acyrthosiphon pisum and named them Bacteriocyte-specific Cysteine-Rich (BCR) peptides; these peptides are exclusively expressed in bacteriocytes, special aphid cells that harbor symbionts. Similar symbiotic organ-specific cysteine-rich peptides identified in the root nodules of leguminous plants are named Nodule-specific Cysteine-Rich (NCR) peptides. NCR peptides target rhizobia in the nodules and are essential for symbiotic nitrogen fixation. A BacA (membrane protein) mutant of Sinorhizobium is sensitive to NCR peptides and is unable to establish symbiosis. Based on the structural and expressional similarities between BCR peptides and NCR peptides, we hypothesized that aphid BCR peptides exhibit antimicrobial activity, similar to some NCR peptides. We herein synthesized BCR peptides and investigated their antimicrobial activities and effects on the bacterial membrane of Escherichia coli. The peptides BCR1, BCR3, BCR5, and BCR8 exhibited antimicrobial activities with increased membrane permeability. An sbmA mutant of E. coli, a homolog of bacA of S. meliloti, was more sensitive to BCR peptides than the wild type. Our results suggest that BCR peptides have properties that may be required to control the endosymbiont, similar to NCR peptides in legumes.

Bacteriocyte Reprogramming to Cope With Nutritional Stress in a Phloem Sap Feeding Hemipteran, the Pea Aphid Acyrthosiphon pisum.

Nutritional symbioses play a central role in the ability of insects to thrive on unbalanced diets and in ensuring their evolutionary success. A genomic model for nutritional symbiosis comprises the hemipteran Acyrthosiphon pisum, and the gamma-3-proteobacterium, Buchnera aphidicola, with genomes encoding highly integrated metabolic pathways. A. pisum feeds exclusively on plant phloem sap, a nutritionally unbalanced diet highly variable in composition, thus raising the question of how this symbiotic system responds to nutritional stress. We addressed this by combining transcriptomic, phenotypic and life history trait analyses to determine the organismal impact of deprivation of tyrosine and phenylalanine. These two aromatic amino acids are essential for aphid development, are synthesized in a metabolic pathway for which the aphid host and the endosymbiont are interdependent, and their concentration can be highly variable in plant phloem sap. We found that this nutritional challenge does not have major phenotypic effects on the pea aphid, except for a limited weight reduction and a 2-day delay in onset of nymph laying. Transcriptomic analyses through aphid development showed a prominent response in bacteriocytes (the core symbiotic tissue which houses the symbionts), but not in gut, thus highlighting the role of bacteriocytes as major modulators of this homeostasis. This response does not involve a direct regulation of tyrosine and phenylalanine biosynthetic pathway and transporter genes. Instead, we observed an extensive transcriptional reprogramming of the bacteriocyte with a rapid down-regulation of genes encoding sugar transporters and genes required for sugar metabolism. Consistently, we observed continued overexpression of the A. pisum homolog of RRAD, a small GTPase implicated in repressing aerobic glycolysis. In addition, we found increased transcription of genes involved in proliferation, cell size control and signaling. We experimentally confirmed the significance of these gene expression changes detecting an increase in bacteriocyte number and cell size in vivo under tyrosine and phenylalanine depletion. Our results support a central role of bacteriocytes in the aphid response to amino acid deprivation: their transcriptional and cellular responses fine-tune host physiology providing the host insect with an effective way to cope with the challenges posed by the variability in composition of phloem sap.

Targeting symbiosis-related insect genes by RNAi in the pea aphid-Buchnera symbiosis.

The growth and reproduction of phloem sap-feeding insects requires the sustained function of intracellular bacteria localized in specialized cells known as bacteriocytes, giving the potential to target the bacterial symbiosis as a novel strategy for controlling sap-feeding insect pests. We focused on two genes in the pea aphid Acyrthosiphon pisum, amiD and ldcA1, which were acquired horizontally from bacteria and have the annotated function to degrade immunogenic bacterial peptidoglycan. We hypothesized that AmiD and LdcA1 function to eliminate peptidoglycan fragments released by the bacterial symbiont Buchnera inhabiting the bacteriocytes, thereby protecting the Buchnera from host attack. Consistent with this hypothesis, expression of amiD and ldcA1 was enriched in bacteriocytes and varied significantly with aphid age, conforming to an inverse curvilinear relationship for amiD and negative linear relationship for ldcA1. RNAi against amiD and ldcA1 administered orally to larval pea aphids caused a significant reduction in Buchnera abundance and activity, accompanied by depressed aphid growth rates. For RNAi experiments, the aphids were co-administered with dsRNA against an aphid nuclease nuc1, protecting the dsRNA against non-specific degradation. These experiments demonstrate that selective suppression of insect symbiosis-related gene function can reduce the performance of an insect pest. Phylogenetic analysis identified amiD and ldcA1 in sequenced genomes of other aphid species, and amiD in related groups of phloem-feeding insects, offering the opportunity for specific controls against a range of insect pests.

Maternal Inheritance of a Single Somatic Animal Cell Displayed by the Bacteriocyte in the Whitefly Bemisia tabaci.

Bacteriocytes are insect cells harboring symbiotic bacteria that are required by their insect host and are transmitted vertically via the female ovary [1]. In most insect groups, the bacteria are released from the bacteriocytes and transferred to the ovary [2, 3], but in whiteflies, maternal bacteriocytes migrate to each egg [4-6], where they have been reported to lyse, releasing the symbionts [1]. To investigate bacteriocyte inheritance in whiteflies further, we applied microsatellite genotyping and genomic analysis to a genetically diverse population of Bemisia tabaci, and we observed the fate of the bacteriocyte in embryos. Surprisingly, the microsatellite profile of the bacteriocytes was uniform, and insect cross experiments demonstrated that the bacteriocytes have a stable genotype that differs from the genotype of the insect head (which lacks bacteriocytes). Comparative genomic analysis indicates that genomes of the bacteriocyte and whitefly head are distinct. Interestingly, the bacterioyte genome contains the canonical arthropod telomere repeats TTAGG, and the bacteriocytes express telomere maintenance genes that may underlie cellular immortality in animal cells [7]. Microscopy observations confirmed that a single bacteriocyte transmitted to each egg is retained and divides once just before egg hatch, yielding two bacteriocytes in the neonate insect. These data demonstrate the maternal inheritance of an absolutely required somatic insect cell, violating the developmental separation of germline and soma [8, 9]. Future investigation on the mechanism and phylogenetic distribution of maternally inherited bacteriocytes will shed light on the developmental origins and evolutionary diversification of bacteriocytes [10] and the processes underlying cellular immortality [11].

An IMD-like pathway mediates both endosymbiont control and host immunity in the cereal weevil Sitophilus spp.

Many insects developing on nutritionally unbalanced diets have evolved symbiotic associations with vertically transmitted intracellular bacteria (endosymbionts) that provide them with metabolic components, thereby improving the host's abilities to thrive on such poor ecological niches. While host-endosymbiont coevolutionary constraints are known to entail massive genomic changes in the microbial partner, host's genomic evolution remains elusive, particularly with regard to the immune system. In the cereal weevil Sitophilus spp., which houses Sodalis pierantonius, endosymbionts are secluded in specialized host cells, the bacteriocytes that group together as an organ, the bacteriome. We previously reported that at standard conditions, the bacteriome highly expresses the coleoptericin A (colA) antimicrobial peptide (AMP), which was shown to prevent endosymbiont escape from the bacteriocytes. However, following the insect systemic infection by pathogens, the bacteriome upregulates a cocktail of AMP encoding genes, including colA. The regulations that allow these contrasted immune responses remain unknown. In this short report, we provide evidence that an IMD-like pathway is conserved in two sibling species of cereal weevils, Sitophilus oryzae and Sitophilus zeamais. RNA interference (RNAi) experiments showed that imd and relish genes are essential for (i) colA expression in the bacteriome under standard conditions, (ii) AMP up-regulation in the bacteriome following a systemic immune challenge, and (iii) AMP systemic induction following an immune challenge. Histological analyses also showed that relish inhibition by RNAi resulted in endosymbiont escape from the bacteriome, strengthening the involvement of an IMD-like pathway in endosymbiont control. We conclude that Sitophilus' IMD-like pathway mediates both the bacteriome immune program involved in endosymbiont seclusion within the bacteriocytes and the systemic and local immune responses to exogenous challenges. This work provides a striking example of how a conserved immune pathway, initially described as essential in pathogen clearance, also functions in the control of mutualistic associations.

Hype or opportunity? Using microbial symbionts in novel strategies for insect pest control.

All insects, including pest species, are colonized by microorganisms, variously located in the gut and within insect tissues. Manipulation of these microbial partners can reduce the pest status of insects, either by modifying insect traits (e.g. altering the host range or tolerance of abiotic conditions, reducing insect competence to vector disease agents) or by reducing fitness. Strategies utilizing heterologous microorganisms (i.e. derived from different insect species) and genetically-modified microbial symbionts are under development, particularly in relation to insect vectors of human disease agents. There is also the potential to target microorganisms absolutely required by the insect, resulting in insect mortality or suppression of insect growth or fecundity. This latter approach is particularly valuable for insect pests that depend on nutrients from symbiotic microorganisms to supplement their nutritionally-inadequate diet, e.g. insects feeding through the life cycle on vertebrate blood (cimicid bugs, anopluran lice, tsetse flies), plant sap (whiteflies, aphids, psyllids, planthoppers, leafhoppers/sharpshooters) and sound wood (various xylophagous beetles and some termites). Further research will facilitate implementation of these novel insect pest control strategies, particularly to ensure specificity of control agents to the pest insect without dissemination of bio-active compounds, novel microorganisms or their genes into the wider environment.

A Novel, Extremely Elongated, and Endocellular Bacterial Symbiont Supports Cuticle Formation of a Grain Pest Beetle.

The saw-toothed grain beetle, Oryzaephilus surinamensis (Silvanidae), is a cosmopolitan stored-product pest. Early studies on O. surinamensis in the 1930s described the presence of peculiar bacteriomes harboring endosymbiotic bacteria in the abdomen. Since then, however, the microbiological nature of the symbiont has been elusive. Here we investigated the endosymbiotic system of O. surinamensis in detail. In the abdomen of adults, pupae, and larvae, four oval bacteriomes were consistently identified, whose cytoplasm was full of extremely elongated tubular bacterial cells several micrometers wide and several hundred micrometers long. Molecular phylogenetic analysis identified the symbiont as a member of the Bacteroidetes, in which the symbiont was the most closely related to the endosymbiont of a grain pest beetle, Rhyzopertha dominica (Bostrichidae). The symbiont was detected in developing embryos, corroborating vertical symbiont transmission through host generations. The symbiont gene showed AT-biased nucleotide composition and accelerated molecular evolution, plausibly reflecting degenerative evolution of the symbiont genome. When the symbiont infection was experimentally removed, the aposymbiotic insects grew and reproduced normally, but exhibited a slightly but significantly more reddish cuticle and lighter body mass. These results indicate that the symbiont of O. surinamensis is not essential for the host's growth and reproduction but contributes to the host's cuticle formation. Symbiont genome sequencing and detailed comparison of fitness parameters between symbiotic and aposymbiotic insects under various environmental conditions will provide further insights into the symbiont's biological roles for the stored-product pest.IMPORTANCE Some beetles notorious as stored-product pests possess well-developed symbiotic organs called bacteriomes for harboring specific symbiotic bacteria, although their biological roles have been poorly understood. Here we report a peculiar endosymbiotic system of a grain pest beetle, Oryzaephilus surinamensis, in which four oval bacteriomes in the abdomen are full of extremely elongated tubular bacterial cells. Experimental symbiont elimination did not hinder the host's growth and reproduction, but resulted in emergence of reddish beetles, uncovering the symbiont's involvement in host's cuticle formation. We speculate that the extremely elongated symbiont cell morphology might be due to the degenerative symbiont genome deficient in bacterial cell division and/or cell wall formation, which highlights an evolutionary consequence of intimate host-symbiont coevolution.