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Psyllids (Hemiptera: Sternorrhyncha: Psylloidea) are plant sap-sucking insects that include important agricultural pests. To obtain insights into the ecological and evolutionary behaviors of microbes, including plant pathogens, in Psylloidea, high-resolution ana-lyses of the microbiomes of nine psyllid species belonging to the family Triozidae were performed using high-throughput amplicon sequencing of the 16S rRNA gene. Analyses identified various bacterial populations, showing that all nine psyllids have at least one secondary symbiont, along with the primary symbiont "Candidatus Carsonella ruddii" (Gammaproteobacteria: Oceanospirillales: Halomonadaceae). The majority of the secondary symbionts were gammaproteobacteria, particularly those of the order Enterobacterales, which included Arsenophonus and Serratia symbiotica, a bacterium formerly recognized only as a secondary symbiont of aphids (Hemiptera: Sternorrhyncha: Aphidoidea). The non-Enterobacterales gammaproteobacteria identified in the present study were Diplorickettsia (Diplorickettsiales: Diplorickettsiaceae), a potential human pathogen, and Carnimonas (Oceanospirillales: Halomonadaceae), a lineage detected for the first time in Psylloidea. Regarding alphaproteobacteria, the potential plant pathogen "Ca. Liberibacter europaeus" (Rhizobiales: Rhizobiaceae) was detected for the first time in Epitrioza yasumatsui, which feeds on the Japanese silverberry Elaeagnus umbellata (Elaeagnaceae), an aggressive invasive plant in the United States and Europe. Besides the detection of Wolbachia (Rickettsiales: Anaplasmataceae) of supergroup B in three psyllid species, a lineage belonging to supergroup O was identified for the first time in Psylloidea. These results suggest the rampant transfer of bacterial symbionts among animals and plants, thereby providing deeper insights into the evolution of interkingdom interactions among multicellular organisms and bacteria, which will facilitate the control of pest psyllids.

Diaphorin is a polyketide produced by Candidatus Profftella armatura (Gammaproteobacteria: Burkholderiales), an obligate symbiont of a notorious agricultural pest, the Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae). Diaphorin belongs to the pederin family of bioactive agents found in various host-symbiont systems, including beetles, lichens, and sponges, harboring phylogenetically diverse bacterial producers. Previous studies showed that diaphorin has inhibitory effects on various eukaryotes, including the natural enemies of D. citri. However, little is known about its effects on prokaryotic organisms. To address this issue, the present study assessed the biological activities of diaphorin on two model prokaryotes, Escherichia coli (Gammaproteobacteria: Enterobacterales) and Bacillus subtilis (Firmicutes: Bacilli). The analyses revealed that diaphorin inhibits the growth of B. subtilis but moderately promotes the growth of E. coli. This finding implies that diaphorin functions as a defensive agent of the holobiont (host + symbionts) against some bacterial lineages but is beneficial for others, which potentially include obligate symbionts of D. citri.ImportanceCertain secondary metabolites, including antibiotics, evolve to mediate interactions among organisms. These molecules have distinct spectra for microorganisms and are often more effective against Gram-positive bacteria than Gram-negative ones. However, it is rare that a single molecule has completely opposite activities on distinct bacterial lineages. The present study revealed that a secondary metabolite synthesized by an organelle-like bacterial symbiont of psyllids inhibits the growth of Gram-positive Bacillus subtilis but promotes the growth of Gram-negative Escherichia coli. This finding not only provides insights into the evolution of symbiosis between animal hosts and bacteria but may also potentially be exploited to promote the effectiveness of industrial material production by microorganisms.

Polyploidy is the state of having multiple copies of the genome within a nucleus or a cell, which has repeatedly evolved across the domains of life. Whereas most bacteria are monoploid, some bacterial species and endosymbiotic organelles that are derived from bacteria are stably polyploid. In the present study, using absolute quantitative PCR, we assessed the ploidy of Candidatus Carsonella ruddii (Gammaproteobacteria, Oceanospirillales), the obligate symbiont of the hackberry petiole gall psyllid, Pachypsylla venusta (Hemiptera, Psylloidea). The genome of this symbiont is one of the smallest known for cellular organisms, at 160 kb. The analysis revealed that

The Asian citrus psyllid Diaphorina citri (Insecta: Hemiptera: Psylloidea), a serious pest of citrus species worldwide, harbors vertically transmitted intracellular mutualists, Candidatus Profftella armatura (Profftella_DC, Gammaproteobacteria: Burkholderiales) and Candidatus Carsonella ruddii (Carsonella_DC, Gammaproteobacteria: Oceanospirillales). Whereas Carsonella_DC is a typical nutritional symbiont, Profftella_DC is a unique defensive symbiont with organelle-like features, including intracellular localization within the host, perfect infection in host populations, vertical transmission over evolutionary time, and drastic genome reduction down to much less than 1 Mb. Large parts of the 460-kb genome of Profftella_DC are devoted to genes for synthesizing a polyketide toxin; diaphorin. To better understand the evolution of this unusual symbiont, the present study analyzed the genome of Profftella_Dco, a sister lineage to Profftella_DC, using Diaphorina cf. continua, a host psyllid congeneric with D. citri. The genome of coresiding Carsonella (Carsonella_Dco) was also analyzed. The analysis revealed nearly perfect synteny conservation in these genomes with their counterparts from D. citri. The substitution rate analysis further demonstrated genomic stability of Profftella which is comparable to that of Carsonella. Profftella_Dco and Profftella_DC shared all genes for the biosynthesis of diaphorin, hemolysin, riboflavin, biotin, and carotenoids, underlining multiple roles of Profftella, which may contribute to stabilizing symbiotic relationships with the host. However, acyl carrier proteins were extensively amplified in polyketide synthases DipP and DipT for diaphorin synthesis in Profftella_Dco. This level of acyl carrier protein augmentation, unprecedented in modular polyketide synthases of any known organism, is not thought to influence the polyketide structure but may improve the synthesis efficiency., Genome Biology and Evolution, 12 (11), ISSN:1759-6653

The Asian citrus psyllid Diaphorina citri (Hemiptera: Psylloidea) is a serious pest of citrus species worldwide because it transmits Candidatus Liberibacter spp. (Alphaproteobacteria: Rhizobiales), the causative agents of the incurable citrus disease, huanglongbing or greening disease. Diaphorina citri possesses a specialized organ called a bacteriome, which harbors vertically transmitted intracellular mutualists, Ca. Carsonella ruddii (Gammaproteobacteria: Oceanospirillales) and Ca. Profftella armatura (Gammaproteobacteria: Betaproteobacteriales). Whereas Carsonella is a typical nutritional symbiont, Profftella is an unprecedented type of toxin-producing defensive symbiont, unusually sharing organelle-like features with nutritional symbionts. Additionally, many D. citri strains are infected with Wolbachia, which manipulate reproduction in various arthropod hosts. In the present study, in an effort to obtain insights into the evolution of symbioses between Diaphorina and bacteria, microbiomes of psyllids closely related to D. citri were investigated. Bacterial populations of Diaphorina cf. continua and Diaphorina lycii were analyzed using Illumina sequencing of 16S rRNA gene amplicons and compared with data obtained from D. citri. The analysis revealed that all three Diaphorina spp. harbor Profftella as well as Carsonella lineages, implying that Profftella is widespread within the genus Diaphorina. Moreover, the analysis identified Ca. Liberibacter europaeus and Diplorickettsia sp. (Gammaproteobacteria: Diplorickettsiales) in D. cf. continua, and a total of four Wolbachia (Alphaproteobacteria: Rickettsiales) lineages in the three psyllid species. These results provide deeper insights into the interactions among insects, bacteria, and plants, which would eventually help to better manage horticulture.

Background Psyllids (Hemiptera: Psylloidea) comprise a group of plant sap-sucking insects that includes important agricultural pests. They have close associations not only with plant pathogens, but also with various microbes, including obligate mutualists and facultative symbionts. Recent studies are revealing that interactions among such bacterial populations are important for psyllid biology and host plant pathology. In the present study, to obtain further insight into the ecological and evolutionary behaviors of bacteria in Psylloidea, we analyzed the microbiomes of 12 psyllid species belonging to the family Psyllidae (11 from Psyllinae and one from Macrocorsinae), using high-throughput amplicon sequencing of the 16S rRNA gene. Results The analysis showed that all 12 psyllids have the primary symbiont, Candidatus Carsonella ruddii (Gammaproteobacteria: Oceanospirillales), and at least one secondary symbiont. The majority of the secondary symbionts were gammaproteobacteria, especially those of the family Enterobacteriaceae (order: Enterobacteriales). Among them, symbionts belonging to “endosymbionts3”, which is a genus-level monophyletic group assigned by the SILVA rRNA database, were the most prevalent and were found in 9 of 11 Psyllinae species. Ca. Fukatsuia symbiotica and Serratia symbiotica, which were recognized only as secondary symbionts of aphids, were also identified. In addition to other Enterobacteriaceae bacteria, including Arsenophonus, Sodalis, and “endosymbionts2”, which is another genus-level clade, Pseudomonas (Pseudomonadales: Pseudomonadaceae) and Diplorickettsia (Diplorickettsiales: Diplorickettsiaceae) were identified. Regarding Alphaproteobacteria, the potential plant pathogen Ca. Liberibacter europaeus (Rhizobiales: Rhizobiaceae) was detected for the first time in Anomoneura mori (Psyllinae), a mulberry pest. Wolbachia (Rickettsiales: Anaplasmataceae) and Rickettsia (Rickettsiales: Rickettsiaceae), plausible host reproduction manipulators that are potential tools to control pest insects, were also detected. Conclusions The present study identified various bacterial symbionts including previously unexpected lineages in psyllids, suggesting considerable interspecific transfer of arthropod symbionts. The findings provide deeper insights into the evolution of interactions among insects, bacteria, and plants, which may be exploited to facilitate the control of pest psyllids in the future.

Diaphorin is a polyketide produced by Candidatus Profftella armatura (Betaproteobacteria), an organelle-like defensive symbiont harbored by a plant sap-sucking insect, Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae). Diaphorin belongs to the pederin family, a group of compounds that share much of their core structure with that of pederin, which is characterized by two dihydropyran rings bridged by an N-acyl aminal. Most members of this family have potent antitumor activity, making them promising anticancer drug candidates. The present study assessed the therapeutic potential of diaphorin for its antitumor activity against 39 human cancer cell lines including those from breast, brain, colon, lung, skin, ovary, kidney, stomach, and prostate. The results showed that diaphorin had inhibitory activity against all 39 cancer cell lines tested. The GI50, TGI, and LC50 values ranged from 0.28 μM- 2.4 μM, 1.6 μM -11 μM, and 7.5 μM-> 100 μM, respectively. These values are among the highest in the pederin family, indicating that the anticancer activity of diaphorin is milder than those of other pederin congeners. The inhibitory effects of diaphorin significantly differed among the distinct cancer types. The maximum difference was about 10-fold, which was similar to those of most other pederin congeners.

Summary Diverse insect species harbor symbiotic bacteria, which play important roles such as provisioning nutrients and providing defense against natural enemies [1–6]. Whereas nutritional symbioses are often indispensable for both partners, defensive symbioses tend to be of a facultative nature [1–12]. The Asian citrus psyllid Diaphorina citri is a notorious agricultural pest that transmits Liberibacter spp. ( Alphaproteobacteria ), causing the devastating citrus greening disease or Huanglongbing [13, 14]. In a symbiotic organ called the bacteriome, D. citri harbors two distinct intracellular symbionts: a putative nutrition provider, Carsonella _DC ( Gammaproteobacteria ), and an unnamed betaproteobacterium with unknown function [15], for which we propose the name " Candidatus Profftella armatura." Here we report that Profftella is a defensive symbiont presumably of an obligate nature with an extremely streamlined genome. The genomes of Profftella and Carsonella _DC were drastically reduced to 464,857 bp and 174,014 bp, respectively, suggesting their ancient and mutually indispensible association with the host. Strikingly, 15% of the small Profftella genome encoded horizontally acquired genes for synthesizing a novel polyketide toxin. The toxin was extracted, pharmacologically and structurally characterized, and designated diaphorin. The presence of Profftella and its diaphorin-biosynthetic genes was perfectly conserved in the world's D. citri populations.

Horizontal gene transfer is the transfer of genetic material across species boundaries. Although horizontal gene transfers are relatively rare in animals, the recent rapid accumulation of genomic data has identified increasing amounts of exogenous DNA inserts in insect genomes. Most of the horizontally acquired sequences appear to be non-functional; however, there is growing evidence that some genes are truly expressed and confer novel functions on the recipient insects. These include previously unavailable metabolic properties including digesting food, degrading toxins, providing resistance to pathogens, and facilitating an obligate mutualistic relationship with intracellular bacteria. A recent analysis revealed that an aphid gene of bacterial origin encodes a protein that is transported into the obligate symbiont, paralleling the evolution of endosymbiotic organelles.

Bacteria confined to intracellular environments experience extensive genome reduction. In extreme cases, insect endosymbionts have evolved genomes that are so gene-poor that they blur the distinction between bacteria and endosymbiotically derived organelles such as mitochondria and plastids. To understand the host’s role in this extreme gene loss, we analyzed gene content and expression in the nuclear genome of the psyllid Pachypsylla venusta, a sap-feeding insect that harbors an ancient endosymbiont (Carsonella) with one of the most reduced bacterial genomes ever identified. Carsonella retains many genes required for synthesis of essential amino acids that are scarce in plant sap, but most of these biosynthetic pathways have been disrupted by gene loss. Host genes that are upregulated in psyllid cells housing Carsonella appear to compensate for endosymbiont gene losses, resulting in highly integrated metabolic pathways that mirror those observed in other sap-feeding insects. The host contribution to these pathways is mediated by a combination of native eukaryotic genes and bacterial genes that were horizontally transferred from multiple donor lineages early in the evolution of psyllids, including one gene that appears to have been directly acquired from Carsonella. By comparing the psyllid genome to a recent analysis of mealybugs, we found that a remarkably similar set of functional pathways have been shaped by independent transfers of bacterial genes to the two hosts. These results show that horizontal gene transfer is an important and recurring mechanism driving coevolution between insects and their bacterial endosymbionts and highlight interesting similarities and contrasts with the evolutionary history of mitochondria and plastids.

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Sternorrhyncha: Psylloidea: Liviidae) is an important pest of citrus species worldwide because it transmits Candidatus Liberibacter spp. (Alphaproteobacteria); the causative agents of an incurable citrus disease known as huanglongbing or greening disease. D. citri possesses a vertically transmitted intracellular symbiont, Candidatus Profftella armatura (Betaproteobacteria), which produces diaphorin; a polyketide that is toxic to various eukaryotic organisms. Our previous study demonstrated that the total amount and average concentration of diaphorin in adult D. citri, when homogeneous distribution is assumed within the insect, are sufficient to exert inhibitory effects on fungi and insects, including the Asian lady beetle Harmonia axyridis (Coleoptera: Coccinellidae); one of the major predators of D. citri. However, diaphorin may be localized to a limited body area within D. citri, and its concentration may change during development. In the present study, to better understand the physiological and ecological function of diaphorin, we assessed the distribution of diaphorin within the D. citri body and analyzed concentrations of diaphorin in various developmental stages. Expression of genes involved in diaphorin synthesis was also analyzed. The results demonstrated that diaphorin is distributed widely in the D. citri body, which appears to be a prerequisite for effective deterrence of natural enemies. The concentration of diaphorin was shown to change significantly during the development of D. citri. It was highest in mature adults, followed by embryos and teneral adults, and lowest in nymphs. The lowest concentrations of diaphorin observed in nymphs are still presumed to be effective in deterring invasive natural enemies, including parasites, parasitoids, and entomopathogenic fungi. Quantitative RT-PCR indicated that amounts of transcripts for diaphorin synthesis genes dipP and dipT were at a minimum in embryos, increased during the nymphal period, and reached a maximum level just after adult eclosion. The alteration pattern of the amounts of transcripts for diaphorin synthesis genes appeared to partially disagree with that of the concentration of diaphorin. The present study provides new insights into the function of diaphorin, which is essential for further investigations that aim to improve the efficacy of D. citri biological control.

The purine salvage pathway recycles purines to nucleotides, promoting efficient utilization of purine nucleotides. Exceptionally among animals with completely sequenced genomes, the pea aphid lacks key purine recycling genes that code for purine nucleoside phosphorylase and adenosine deaminase, indicating that the aphid can neither metabolize nucleosides to the corresponding purines, nor adenosine to inosine. Purine metabolism genes in the symbiotic bacterium Buchnera complement aphid genes, and Buchnera can meet its nucleotide requirement from aphid-derived guanosine. Buchnera demand for nucleosides may have relaxed the selection for purine recycling in the aphid, leading to the loss of key aphid purine salvage genes. Further, the coupled purine metabolism of aphid and Buchnera could contribute to the dependence of the pea aphid on this symbiosis.

In insects, chitinases play an essential role in the degradation of old exoskeleton and turnover of the gut lining. In silico screening of the entire genome of the pea aphid (Hemimetabola), Acyrthosiphon pisum, detected nine genes encoding putative chitinase-like proteins, including six enzymatically active chitinases, one imaginal disc growth factor, and one endo-beta-N-acetylglucosaminidase. Screening of the genomes of Aedes aegypti, Anopheles gambiae, Apis mellifera, Bombyx mori, Culex quinquefasciatus, Drosophila melanogaster, Nasonia vitripennis, Pediculus humanus corporis, and Tribolium castaneum suggested repeated gene duplications in holometabolous lineages. Quantitative reverse transcription-PCR demonstrated the expression of four and two distinct chitinase-like genes of A. pisum to be highly up-regulated in the embryo and the midgut, respectively, suggesting specific roles in these pea aphid tissues.

Aphids are the primary vectors of plant viruses. Transmission can occur via attachment to the cuticle lining of the insect (non-circulative transmission) or after internalization in the insect cells with or without replication (circulative transmission). In this paper, we have focused on the circulative and non-propagative mode during which virions enter the cell following receptor-mediated endocytosis, are transported across the cell in vesicles and released by exocytosis without replicating. The correct uptake, transport and delivery of the vesicles cargo relies on the participation of proteins from different families which have been identified in the Acyrthosiphon pisum genome. Assemblage of this annotated dataset provides a useful basis to improve our understanding of the molecules and mechanisms involved in virus transmission by A. pisum and other aphid species.

Screening of the entire genome of the pea aphid, Acyrthosiphon pisum, detected 15 genes for putative dynamin superfamily proteins - self-assembling large GTPases that are involved in the fission and fusion of membranes. In addition to a single gene each for Dyn, Drp1, and Opa1, orthologues that are common in Metazoa, 12 genes encoding a novel type of dynamin were found. Phylogenetic analyses showed that these novel-class genes are monophyletic. Quantitative reverse transcription-PCR demonstrated that expressions of four novel-class dynamin genes are highly up-regulated in the midgut, through which aphids take in phloem-sap diets and plant viruses. As this type of dynamin is absent from all other fully sequenced organisms, they may function in processes unique to aphids.

Large collections of full-length cDNAs are important resources for genome annotation and functional genomics. We report the creation of a collection of 50 599 full-length cDNA clones from the pea aphid, Acyrthosiphon pisum. Sequencing from 5′ and 3′ ends of the clones generated 97 828 high-quality expressed sequence tags, representing approximately 9000 genes. These sequences were imported to AphidBase and are shown to play crucial roles in both automatic gene prediction and manual annotation. Our detailed analyses demonstrated that the full-length cDNAs can further improve gene models and can even identify novel genes that are not included in the current version of the official gene set. This full-length cDNA collection can be utilized for a wide variety of functional studies, serving as a community resource for the study of the functional genomics of the pea aphid.

The Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a serious pest worldwide, transmitting Candidatus Liberibacter spp. (Alphaproteobacteria), the causative agents of a devastating citrus disease known as huanglongbing or greening disease. In a symbiotic organ called the bacteriome, D. citri possesses an organelle-like defensive symbiont, Candidatus Profftella armatura (Betaproteobacteria), and a nutritional symbiont, Ca. Carsonella ruddii (Gammaproteobacteria). Drastically reduced symbiont genomes and metabolic complementarity among the symbionts and D. citri indicate their mutually indispensable association. Moreover, horizontal gene transfer between the Profftella and Liberibacter lineages suggests ecological and evolutionary interactions between the bacteriome symbiont and the HLB pathogen. Using fluorescence in situ hybridization, we examined the behavior of Profftella and Carsonella during transovarial transmission and the development of D. citri. In the bacteriomes of sexually-mature female adults, symbionts transformed from an extremely elongated tubular form into spherical or short-rod forms, which migrated toward the ovary. The symbionts then formed mosaic masses, which entered at the posterior pole of the vitellogenic oocytes. After anatrepsis, Carsonella and Profftella migrated to the central and peripheral parts of the mass, respectively. Following the appearance of host nuclei, the mass cellularized, segregating Carsonella and Profftella in the central syncytium and peripheral uninucleate bacteriocytes, respectively. Subsequently, the uninucleate bacteriocytes harboring Profftella assembled at the posterior pole, while the syncytium, containing Carsonella, sat on the anterior side facing the germ band initiating katatrepsis. During dorsal closure, the syncytium was divided into uninuclear bacteriocytes, which surrounded the mass of bacteriocytes containing Profftella. Once fully surrounded, the bacteriocyte mass containing Profftella was fused into a syncytium. Prior to hatching, a pair of wing-like protrusions arose from both lateral sides of the bacteriome, which continued to grow throughout the nymphal stages. These findings provide a foundation for better understanding the intricate relationship between D. citri and its microbiota.

Aphids possess bacteriocytes, cells specifically differentiated to harbor obligatory mutualistic bacteria of the genus Buchnera , which have lost many genes that are essential for common bacterial functions. To understand the host's role in maintaining the symbiotic relationship, bacteriocytes were isolated from the pea aphid, Acyrthosiphon pisum , and the host transcriptome was investigated by using EST analysis and real-time quantitative RT-PCR. A number of genes were highly expressed specifically in the bacteriocyte, including ( i ) genes for amino acid metabolism, including those for biosynthesis of amino acids that Buchnera cannot produce, and those for utilization of amino acids that Buchnera can synthesize; ( ii ) genes related to transport, including genes for mitochondrial transporters and a gene encoding Rab, a G protein that regulates vesicular transport; and ( iii ) genes for putative lysozymes that degrade bacterial cell walls. Significant up-regulation of i clearly indicated that the bacteriocyte is involved in the exchange of amino acids between the host aphid and Buchnera , the key metabolic process in the symbiotic system. Conspicuously high expression of ii and iii shed light on previously unknown aspects of the host– Buchnera interactions in the symbiotic system.

Aposymbiotic pea aphids, which were deprived of their intracellular symbiotic bacterium, Buchnera, exhibit growth retardation and no fecundity. High performance liquid chromatographic (HPLC) analysis revealed that these aposymbiotic aphids, when reared on broad bean plants, accumulated a large amount of histamine. To assess the possibility of extraordinary proliferation of microorganisms other than Buchnera, we enumerated eubacteria and fungi in aphids using the real-time quantitative PCR method that targets genes encoding small-subunit rRNAs. The result showed that these microorganisms were extremely abundant in the aposymbiotic aphids reared on plants. Microbial communities in aposymbiotic aphids were further profiled by phylogenetic analysis of small-subunit rDNAs. Of 172 nonchimeric sequences of fungal 18S rDNAs, 138 (80.2%) belonged to the phylum Ascomycota. Among them, 21 clustered within a monophyletic group consisting of insect-pathogenic fungi and yeast-like symbionts of homopteran insects. Thirty-one (18.0%), two (1.2%), and one (0.6%) clones were clustered within the Basidiomycota, Zygomycota, and Oomycota, respectively. Of 167 nonchimeric sequences of eubacterial 16S rDNAs, 84 (50.3%) belonged to the gamma-subdivision of Proteobacteria to which most primary endosymbionts of insects and prolific histamine producers belong. Forty (24.0%), 25 (15.0%), 10 (6.0%), and five (3.0%) clones were clustered within alpha-Proteobacteria, Cytophaga-Flavobacterium-Bacteroides (CFB) group, Actinobacteria, and beta-Proteobacteria, respectively. Three had no phylogenetic association with known taxonomic divisions. None of the sequences studied in this study coincided exactly with those deposited in GenBank.

Summary During the course of the evolution of endosymbiotic organelles, mitochondria and plastids, numerous genes were transferred from ancestral organelles and other bacteria to the host genome. This process required, firstly, incorporating DNA fragments encoding intact genes into the host genome, secondly, acquiring the expression signals such as eukaryotic promoters and regulatory elements that enable their transcription in eukaryotic hosts, and thirdly, in many cases, evolving a dedicated targeting system to transport and import their protein products into the endosymbiotic organelles [1]. The advent of this protein-targeting machinery is commonly assumed to be the most crucial step, at which point an endosymbiont becomes an organelle [2,3]. Bacterial lineages have repeatedly evolved intimate symbioses with eukaryotic hosts, but the establishment of the protein translocation system has been shown only in the cases of bona fide organelles and the photosynthetic symbiosis in a cercozoan amoeba [3]. Here, we report that an aphid gene that was horizontally acquired from a bacterium produces protein, which is transported to an obligate intracellular bacterial symbiont. To our knowledge, this is the first report of integration between multicellular eukaryotes and bacteria to the extent of ‘organellogenesis'.

Differential cDNA display and quantitative RT-PCR revealed that mRNA of host S-adenosylmethionine decarboxylase (SAMDC) was abundant only in the aphid endosymbiotic system well organized in young hosts, suggesting that SAMDC plays some important roles in the system. SAMDC is a key enzyme to synthesize polyamines that are known to be involved in a large array of biological events including protein synthesis, DNA stabilization, DNA replication, and cell proliferation. As the first step to investigate roles of polyamines in the endosymbiotic system, polyamine composition in bacteriocytes was determined by high performance liquid chromatography. As a result, we found that bacteriocytes contained virtually an only single polyamine, spermidine. The spermidine content of bacteriocytes fluctuated with time in the course of development and aging of the host aphid. This is the first report of polyamine assessment in a prokaryote‐eukaryote endocellular symbiotic system, which demonstrated a unique polyamine composition. © 2001 Elsevier Science Ltd. All rights reserved.

he Asian citrus psyllid Diaphorina citri is a notorious agricultural pest that transmits the phloem-inhabiting alphaproteobacterial ‘Candidatus Liberibacter asiaticus’ and allied plant pathogens, which cause the devastating citrus disease called Huanglongbing or greening disease. D. citri harbors two distinct bacterial mutualists in the symbiotic organ called bacteriome: the betaproteobacterium ‘Candidatus Profftella armatura’ in the syncytial cytoplasm at the center of the bacteriome, and the gammaproteobacterium ‘Candidatus Carsonella ruddii’ in uninucleate bacteriocytes. Here we report that a putative amino acid transporter LysE of Profftella forms a highly supported clade with proteins of L. asiaticus, L. americanus, and L. solanacearum. L. crescens, the most basal Liberibacter lineage currently known, lacked the corresponding gene. The Profftella-Liberibacter subclade of LysE formed a clade with proteins from betaproteobacteria of the order Burkholderiales, to which Profftella belongs. This phylogenetic pattern favors the hypothesis that the Liberibacter lineage acquired the gene from the Profftella lineage via horizontal gene transfer (HGT) after L. crescens diverged from other Liberibacter lineages. K A/K S analyses further supported the hypothesis that the genes encoded in the Liberibacter genomes are functional. These findings highlight the possible evolutionary importance of HGT between plant pathogens and their insect vector’s symbionts that are confined in the symbiotic organ and seemingly sequestered from external microbial populations.

Components of the uterine fluid in mice with experimentally-induced adenomyosis and in controls were examined by proton nuclear magnetic resonance spectroscopy. One of the components was markedly different in mice with adenomyosis. As this component was estimated to be citrate by comparison with authentic samples (standard spectrum), its levels in uterine fluid, uterine tissue and blood were determined by enzymatic analysis. The fluid obtained from the uterus with adenomyosis showed significantly lower concentration of citrate than that from normal uterus. However, the uterine tissue concentration was significantly higher in the experimental mice with adenomyosis. There was no difference in the blood citrate level between the experimental and the control groups. Since adenomyosis was induced by chronic hyperprolactinemia, the change of citrate level in the uterus with this lesion might imply some effects of prolactin (PRL) on metabolism and/or secretion of citrate. However, in normal mice, no significant change was demonstrated in uterine citrate concentration after short-term experimental modulation of the circulating PRL level. Thus, it is unlikely that PRL can regulate directly citrate metabolism in the uterus, indicating some other cause for changes in citrate level accompanying the development of adenomyosis.

Genome reduction is typical of obligate symbionts. In cellular organelles, this reduction partly reflects transfer of ancestral bacterial genes to the host genome, but little is known about gene transfer in other obligate symbioses. Aphids harbor anciently acquired obligate mutualists, Buchnera aphidicola (Gammaproteobacteria), which have highly reduced genomes (420–650 kb), raising the possibility of gene transfer from ancestral Buchnera to the aphid genome. In addition, aphids often harbor other bacteria that also are potential sources of transferred genes. Previous limited sampling of genes expressed in bacteriocytes, the specialized cells that harbor Buchnera, revealed that aphids acquired at least two genes from bacteria. The newly sequenced genome of the pea aphid, Acyrthosiphon pisum, presents the first opportunity for a complete inventory of genes transferred from bacteria to the host genome in the context of an ancient obligate symbiosis. Computational screening of the entire A. pisum genome, followed by phylogenetic and experimental analyses, provided strong support for the transfer of 12 genes or gene fragments from bacteria to the aphid genome: three LD–carboxypeptidases (LdcA1, LdcA2,ψLdcA), five rare lipoprotein As (RlpA1-5), N-acetylmuramoyl-L-alanine amidase (AmiD), 1,4-beta-N-acetylmuramidase (bLys), DNA polymerase III alpha chain (ψDnaE), and ATP synthase delta chain (ψAtpH). Buchnera was the apparent source of two highly truncated pseudogenes (ψDnaE and ψAtpH). Most other transferred genes were closely related to genes from relatives of Wolbachia (Alphaproteobacteria). At least eight of the transferred genes (LdcA1, AmiD, RlpA1-5, bLys) appear to be functional, and expression of seven (LdcA1, AmiD, RlpA1-5) are highly upregulated in bacteriocytes. The LdcAs and RlpAs appear to have been duplicated after transfer. Our results excluded the hypothesis that genome reduction in Buchnera has been accompanied by gene transfer to the host nuclear genome, but suggest that aphids utilize a set of duplicated genes acquired from other bacteria in the context of the Buchnera–aphid mutualism., Author Summary Bacterial lineages have repeatedly evolved intimate symbioses with eukaryotic hosts, the most famous cases being those of the cell organelles, mitochondria, and plastids. Symbiont genomes typically lose many ancestral genes, raising the question of how they function with so few genes. In organelles, part of the answer involves gene transfer to the host genome, allowing maintenance of essential functions. So far, the extent of gene transfer to hosts has not been assessed for other cases of intimate, obligate symbiosis. Aphids harbor an ancient coevolved intracellular symbiont, called Buchnera. We used the newly available sequence of the pea aphid genome to conduct an exhaustive computational search for genes of bacterial ancestry. We found that no functional genes have been transferred from Buchnera, ruling out such transfer as a driving force in genome reduction in this symbiont. However, the aphid genome does contain eight transcribed genes of apparent bacterial origin, some of which have been duplicated after transfer. Based on their expression patterns, most of these appear to function specifically in the aphid-Buchnera symbiosis, presenting the possibility that the maintenance of obligate intracellular symbioses can be affected by the acquisition and duplication of genes transferred from unrelated bacterial lineages.

Background Recent genomic analyses of arthropod defense mechanisms suggest conservation of key elements underlying responses to pathogens, parasites and stresses. At the center of pathogen-induced immune responses are signaling pathways triggered by the recognition of fungal, bacterial and viral signatures. These pathways result in the production of response molecules, such as antimicrobial peptides and lysozymes, which degrade or destroy invaders. Using the recently sequenced genome of the pea aphid (Acyrthosiphon pisum), we conducted the first extensive annotation of the immune and stress gene repertoire of a hemipterous insect, which is phylogenetically distantly related to previously characterized insects models. Results Strikingly, pea aphids appear to be missing genes present in insect genomes characterized to date and thought critical for recognition, signaling and killing of microbes. In line with results of gene annotation, experimental analyses designed to characterize immune response through the isolation of RNA transcripts and proteins from immune-challenged pea aphids uncovered few immune-related products. Gene expression studies, however, indicated some expression of immune and stress-related genes. Conclusions The absence of genes suspected to be essential for the insect immune response suggests that the traditional view of insect immunity may not be as broadly applicable as once thought. The limitations of the aphid immune system may be representative of a broad range of insects, or may be aphid specific. We suggest that several aspects of the aphid life style, such as their association with microbial symbionts, could facilitate survival without strong immune protection., Genome Biology, 11 (2), ISSN:1474-760X

Background Aphids possess bacteriocytes, which are cells specifically differentiated to harbour the obligate mutualist Buchnera aphidicola (γ-Proteobacteria). Buchnera has lost many of the genes that appear to be essential for bacterial life. From the bacteriocyte of the pea aphid Acyrthosiphon pisum, we previously identified two clusters of expressed sequence tags that display similarity only to bacterial genes. Southern blot analysis demonstrated that they are encoded in the aphid genome. In this study, in order to assess the possibility of lateral gene transfer, we determined the full-length sequences of these transcripts, and performed detailed structural and phylogenetic analyses. We further examined their expression levels in the bacteriocyte using real-time quantitative RT-PCR. Results Sequence similarity searches demonstrated that these fully sequenced transcripts are significantly similar to the bacterial genes ldcA (product, LD-carboxypeptidase) and rlpA (product, rare lipoprotein A), respectively. Buchnera lacks these genes, whereas many other bacteria, including Escherichia coli, a close relative of Buchnera, possess both ldcA and rlpA. Molecular phylogenetic analysis clearly demonstrated that the aphid ldcA was derived from a rickettsial bacterium closely related to the extant Wolbachia spp. (α-Proteobacteria, Rickettsiales), which are intracellular symbionts of various lineages of arthropods. The evolutionary origin of rlpA was not fully resolved, but it was clearly demonstrated that its double-ψ β-barrel domain is of bacterial origin. Real-time quantitative RT-PCR demonstrated that ldcA and rlpA are expressed 11.6 and 154-fold higher in the bacteriocyte than in the whole body, respectively. LdcA is an enzyme required for recycling murein (peptidoglycan), which is a component of the bacterial cell wall. As Buchnera possesses a cell wall composed of murein but lacks ldcA, a high level of expression of the aphid ldcA in the bacteriocyte may be essential to maintain Buchnera. Although the function of RlpA is not well known, conspicuous up-regulation of the aphid rlpA in the bacteriocyte implies that this gene is also essential for Buchnera. Conclusion In this study, we obtained several lines of evidence indicating that aphids acquired genes from bacteria via lateral gene transfer and that these genes are used to maintain the obligately mutualistic bacterium, Buchnera.

Insect heritable symbionts have proven to be ubiquitous, based on molecular screening of various insect lineages. Recently, molecular and experimental approaches have yielded an immensely richer understanding of their diverse biological roles, resulting in a burgeoning research literature. Increasingly, commonalities and intermediates are being discovered between categories of symbionts once considered distinct: obligate mutualists that provision nutrients, facultative mutualists that provide protection against enemies or stress, and symbionts such as Wolbachia that manipulate reproductive systems. Among the most far-reaching impacts of widespread heritable symbiosis is that it may promote speciation by increasing reproductive and ecological isolation of host populations, and it effectively provides a means for transfer of genetic information among host lineages. In addition, insect symbionts provide some of the extremes of cellular genomes, including the smallest and the fastest evolving, raising new questions about the limits of evolution of life.

Previous studies have suggested that the minimal cellular genome could be as small as 400 kilobases. Here, we report the complete genome sequence of the psyllid symbiont Carsonella ruddii, which consists of a circular chromosome of 159,662 base pairs, averaging 16.5% GC content. It is by far the smallest and most AT-rich bacterial genome yet characterized. The genome has a high coding density (97%) with many overlapping genes and reduced gene length. Genes for translation and amino acid biosynthesis are relatively well represented, but numerous genes considered essential for life are missing, suggesting that Carsonella may have achieved organelle-like status.

Polyamine composition in an aphid endosymbiotic bacterium, Buchnera sp., was determined by high-performance liquid chromatographic analysis. We found that Buchnera contained virtually only a single polyamine, spermidine. The spermidine content of Buchnera was considerably higher in young aphids and tended to decrease with the age of the host. Expression of speD and speE , whose gene products are key enzymes in the synthesis of spermidine, was analyzed by real-time quantitative reverse transcription-PCR. It was shown that the levels of their mRNAs fluctuated in line with the spermidine content.

Bacteriocytes harbouring Buchnera endosymbionts were isolated from young and old aphids, Acyrthosiphon pisum, and their mRNA populations were examined by the differential cDNA display and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) technique. It was suggested that several gene products are produced when the symbiotic system is well organized in the young insect, whereas others are produced after the system becomes degenerate in old aphids. Among the gene products that were actively synthesized in the symbiotic system of the young host were putative aspartate aminotransferase, homoserine kinase and N-acetylglutamate synthase. These findings were consistent with the hypothesis that the symbiotic system utilizes glutamate to produce essential amino acids.

Aphids are the leading pests in agricultural crops. A large-scale sequencing of 40,904 ESTs from the pea aphid Acyrthosiphon pisum was carried out to define a catalog of 12,082 unique transcripts. A strong AT bias was found, indicating a compositional shift between Drosophila melanogaster and A. pisum. An in silico profiling analysis characterized 135 transcripts specific to pea-aphid tissues (relating to bacteriocytes and parthenogenetic embryos). This project is the first to address the genetics of the Hemiptera and of a hemimetabolous insect.