Codon usage bias and tRNA over-expression in Buchnera aphidicola after aromatic amino acid nutritional stress on its host Acyrthosiphon pisum

Codon usage bias and relative abundances of tRNA isoacceptors were analysed in the obligate intracellular symbiotic bacterium, Buchnera aphidicola from the aphid Acyrthosiphon pisum, using a dedicated 35mer oligonucleotide microarray. Buchnera is archetypal of organisms living with minimal metabolic requirements and presents a reduced genome with high-evolutionary rate. Codonusage in Buchnera has been overcome by the high mutational bias towards AT bases. However, several lines of evidence for codon usage selection are given here. A significant correlation was found between tRNA relative abundances and codon composition of Buchnera genes. A significant codon usage bias was found for the choice of rare codons in Buchnera: C-ending codons are preferred in highly expressed genes, whereas G-ending codons are avoided. This bias is not explained by GC skew in the bacteria and might correspond to a selection for perfect matching between codon–anticodon pairs for some essential amino acids in Buchnera proteins. Nutritional stress applied to the aphid host induced a significant overexpression of most of the tRNA isoacceptors in bacteria. Although, molecular regulation of the tRNA operons in Buchnera was not investigated, a correlation between relative expression levels and organization in transcription unit was found in the genome of Buchnera

Analyse par digitalisation d'images du comportement d'affouragement d'une fourmi attine : application aux tests de choix

Nous décrivons l’automatisation de l’enregistrement et de l’analyse du comportement d’affouragement d’une fourmi attine Acromyrmex octospinosus (Reich) en vue de diverses applications à des tests de préférence. Le montage est réalisé à l’aide d’un micro-ordinateur couplé à une caméra électronique. Le comportement analysé est la prise de confetti (de limbe de différentes espèces végétales ou de papier filtre imbibé d’extraits à éprouver) déposés au hasard sur une grille de choix. Les avantages et les limites de cette méthode sont discutés.A new method is described for the automatic recording and analysis of foraging behaviour in the leaf-cutting ant Acromyrmex octospinosus (Reich). This method had various applications in relation to preference tests. The setup involved a micro-computer linked to a video camera. The behaviour analysed was selection of "confetti" (leaf-discs cut from various plant species or filter-paper discs treated with extracts to be tested) placed randomly on a grid. The advantages and limitations of this method are discussed

Isolement, caractérisation et cibles de nouveaux inhibiteurs de protéases pour la création de plantes transgéniques résistantes aux pucerons

Parmi les insectes phytophages, les pucerons sont particuliers car ils se nourrissent de sève élaborée. Leurs pièces buccales leur permettant d'effectuer des piqûres dans la plante et d' atteindre les faisceaux du phloème, un compartiment dont le ratio protéines/acides aminés est très déséquilibré. C'est pourquoi, dans ce contexte, les pucerons sont réputés comme ne possédant pas l'arsenal enzymatique permettant une bonne utilisation des protéines. Aussi les stratégies utilisant les inhibiteurs de protéases (IP) comme polypeptide entomotoxique ne semblent pas adaptées a priori à ce groupe d'insectes. Or plusieurs IP ont montré des toxicités paradoxales contre les pucerons. Cette étude a pour objectif d'accroître la disponibilité des gènes d'IP originaux et d'en comprendre le mode d'action afin d'en maîtriser l'introduction raisonnée dans les programmes de lutte variétale. Au cours de ce travail de thèse, des informations sur la protéolyse digestive des Homoptères ont été apportées: des protéases à cystéine, les cathepsines (enzymes lysosomiales) sont spécifiquement exprimées dans le tube digestif et dans un organe spécialisé, le bactériocyte. Elles sont la cible potentielle d'inhibiteurs de protéases à cystéine. Lorsque l'Oryzacystatine (inhibiteur de protéase à cystéine, isolé du grain de riz) est exprimé dans le colza, la fécondité du puceron Mysus persicae diminue de 25%. Ce résultat démontre qu'il est donc possible d'exprimer des IP dans le phloème pour lutter contre les pucerons. L'amélioration, par mutagenèse dirigée, d'un inhibiteur de protéases à sérine, isolé du pois, a aussi été effectuée mais son expression hétérologue dans Pichia pastoris ou Arabidospsis thaliana n'a pas permis d'obtenir une forme active. Nous avons cherché à élargir le pool de gènes disponible pour lutter contre les pucerons. L'isolement d'un inhibiteur de protéases à cystéine de l'hémolymphe du puceron a été entreprise au niveau moléculaire et biochimique. Ces résultats démontrent que les IP constituent une nouvelle voie pour lutter contre les pucerons.Among the phytophagous insects, aphids show the particularity of feeding on sap. Their stylets allow them to reach the phloem tissues, a compartment which protein/amino acid ration is low. In this context, aphids are considered to be devoid of the enzymatic stock capable of hydrolysing protein. Strategies using proteinase inhibitor (PI) as entomotoxic polypeptide are considered at first inadequate to fight this kind of insects. Nevertheless, several PI have paradox toxicity against aphids. This study is aimed at increasing the number of potential novel PI, at understanding their function in order to introduce them in biologic fight program. During this work, new informations on digestive proteolysis in Homoptera were gained: cystein proteinase cathepsins (lysosomal enzyme) are specifically expressed in gut and in a specialized organ, the bacteriocyte. They are potential targets of cystein-PI. Seedrapes expressing Oryzacystatine (isolated from rice seed) induce a 25% reduction of Myzus persicea fecundity. This result shows the possibility to express PI in phloem to fight aphids. Improvement of a serine-PI (isolated from pea) via directed mutagenesis was performed but no active form was obtained neither in Pichia pastoris nor in Arabidosis thaliana expression system. We tried to enlarge the gene pool to fight aphids. Isolation of a new cystein-PI from aphid haemolymph was initiated at the molecular and biochemical level. These results show that PI constitute a new strategy to fight aphids.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

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.status: publishe

Conservation of the links between gene transcription and chromosomal organization in the highly reduced genome of -1

<p><b>Copyright information:</b></p><p>Taken from "Conservation of the links between gene transcription and chromosomal organization in the highly reduced genome of "</p><p>http://www.biomedcentral.com/1471-2164/8/143</p><p>BMC Genomics 2007;8():143-143.</p><p>Published online 4 Jun 2007</p><p>PMCID:PMC1899503.</p><p></p>r not ("nOp"). The error bars indicate the 95% confidence interval of the mean. After applying the Bonferroni correction for multiple tests, the significant differences obtained by ANOVA and Likelihood ratio tests between two classes of genes are marked with the same symbol: * significant difference between "Op" and "nOp" for "E"; † significant difference between "Op" and "nOp" for "nE"; ‡ significant difference between "E" and "nE" for "Op"; no significant difference was observed between "E" and "nE" for "nOp"

Conservation of the links between gene transcription and chromosomal organization in the highly reduced genome of -0

<p><b>Copyright information:</b></p><p>Taken from "Conservation of the links between gene transcription and chromosomal organization in the highly reduced genome of "</p><p>http://www.biomedcentral.com/1471-2164/8/143</p><p>BMC Genomics 2007;8():143-143.</p><p>Published online 4 Jun 2007</p><p>PMCID:PMC1899503.</p><p></p>3-probe genes of the oligo-array are reported here. Genes are ranged by increasing differences in standard deviation. The 34 genes of the yellow area (positive differences) show a reduction of their probe standard deviation after gDNA normalization