A full-length cDNA resource for the pea aphid, Acyrthosiphon pisum.

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.

Expansion of genes encoding a novel type of dynamin in the genome of the pea aphid, Acyrthosiphon pisum.

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.

Chitinase-like proteins encoded in the genome of the pea aphid, Acyrthosiphon pisum.

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.

Genomic evidence for complementary purine metabolism in the pea aphid, Acyrthosiphon pisum, and its symbiotic bacterium Buchnera aphidicola.

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.

A genomic analysis of transcytosis in the pea aphid, Acyrthosiphon pisum, a mechanism involved in virus transmission.

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.

Genome size of Pachypsylla venusta (Hemiptera: Psyllidae) and the ploidy of its bacteriocyte, the symbiotic host cell that harbors intracellular mutualistic bacteria with the smallest cellular genome.

Psyllids harbor the primary symbiont, Carsonella ruddii (gamma-Proteobacteria), within the cytoplasm of specialized cells called bacteriocytes. Carsonella has the smallest known cellular genome (160 kb), lacking numerous genes that appear to be essential for bacterial life. This raises the question regarding the genetic mechanisms of the host which supports the survival of Carsonella. Our preceding analyses have indicated that some of the genes that are encoded in the psyllid genome and which are highly expressed in the bacteriocyte are of bacterial origin. This implies that psyllids acquired genes from bacteria by lateral gene transfer (LGT) and are using these genes to maintain the primary symbiont, Carsonella. To reveal the complete picture of LGT from symbiotic bacteria to the genome of psyllids, whole genome analysis of psyllids is essential. In order to assess the feasibility of whole genome analysis of the host psyllid, the genome size of the hackberry petiole gall psyllid, Pachypsylla venusta, was estimated. Feulgen image analysis densitometry and flow cytometry demonstrated that the haploid genome size of P. venusta is 0.74 pg (724 Mb), verifying the feasibility of whole genome analysis. Feulgen image analysis densitometry further revealed that bacteriocytes of P. venusta are invariably 16-ploid. This higher ploidy may be essential to facilitate the symbiotic relationship with bacteria, as it appears to be a feature common to insect bacteriocytes. These results provide a foundation for genomics-based research into host-symbiont interactions.

Expression of host S-adenosylmethionine decarboxylase gene and polyamine composition in aphid bacteriocytes.

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.

Polyamine composition and expression of genes related to polyamine biosynthesis in an aphid endosymbiont, Buchnera.

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.

Provision of riboflavin to the host aphid, Acyrthosiphon pisum, by endosymbiotic bacteria, Buchnera.

Differential cDNA display and quantitative RT-PCR suggested that the riboflavin synthase complex of the aphid endosymbiont, Buchnera, is active only when the symbiotic system is maintained and well organized in young hosts. Since this finding suggested the provision of riboflavin by Buchnera, we examined the effect of dietary riboflavin on the performance of symbiotic and aposymbiotic aphids using chemically-defined diets. Our results indicate: (1) dietary riboflavin is slightly detrimental to young, symbiotic aphids; (2) dietary riboflavin is essential to aposymbiotic aphids; (3) dietary riboflavin remarkably improves the performance of aposymbiotic aphids. These results strongly suggest that young, symbiotic aphids are provided with riboflavin by their endosymbionts, Buchnera.

Differential display of mRNAs related to amino acid metabolism in the endosymbiotic system of aphids.

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.

Changes in citrate concentration in the mouse uterus with experimentally-induced adenomyosis.

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.