Gene knockdown by RNAi in the pea aphid Acyrthosiphon pisum

BackgroundRNA interference (RNAi) is a powerful method to inhibit gene expression in a sequence specific manner.ResultsHere, we described the development of RNAi by micro-injection of double-stranded RNA (dsRNA) in the pea aphid Acyrthosiphon pisum. Injection of dsRNA into whole aphid body induced the silencing of two marker genes with different expression patterns: the ubiquitously expressed Ap-crt genes encoding a calreticulin and the gut specific Ap-cath-L gene encoding a cathepsin-L. Time-course analysis of the silencing showed similar temporal patterns for both genes: inhibition started at 1 day after injection, reached its maximum at 5 days and stopped at 7 days. A comparable 40% decrease of gene expression was observed for Ap-crt and Ap-cath-L.ConclusionThe pea aphid is the first Hemipteran insect for which genome sequence will be available soon. The gene knockdown technique developed in this study will be an essential post-genomic tool for further investigations in aphidology.

[1]  K. Ober,et al.  The roles of wingless and decapentaplegic in axis and appendage development in the red flour beetle, Tribolium castaneum. , 2006, Developmental biology.

[2]  L. Jouanin,et al.  Effects of the cysteine protease inhibitor oryzacystatin (OC-I) on different aphids and reduced performance of Myzus persicae on OC-I expressing transgenic oilseed rape , 2003 .

[3]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[4]  Fabrice Legeai,et al.  AphidBase: a database for aphid genomic resources , 2007, Bioinform..

[5]  Y. Tomoyasu,et al.  Larval RNAi in Tribolium (Coleoptera) for analyzing adult development , 2004, Development Genes and Evolution.

[6]  X. Belles,et al.  Functions of the ecdysone receptor isoform-A in the hemimetabolous insect Blattella germanica revealed by systemic RNAi in vivo. , 2006, Developmental biology.

[7]  R. Araujo,et al.  RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection. , 2006, Insect biochemistry and molecular biology.

[8]  J. Sauer,et al.  RNA interference in ticks: a study using histamine binding protein dsRNA in the female tick Amblyomma americanum , 2003, Insect molecular biology.

[9]  G. Reeck,et al.  RNAi Knockdown of a Salivary Transcript Leading to Lethality in the Pea Aphid, Acyrthosiphon pisum , 2006, Journal of insect science.

[10]  E. Levashina,et al.  Silencing of Toll pathway components by direct injection of double-stranded RNA into Drosophila adult flies. , 2003, Nucleic acids research.

[11]  A. Moya,et al.  Identification of a gene overexpressed in aphids reared under short photoperiod. , 2003, Insect biochemistry and molecular biology.

[12]  R. Newcomb,et al.  RNA interference in the light brown apple moth, Epiphyas postvittana (Walker) induced by double‐stranded RNA feeding , 2006, Insect molecular biology.

[13]  H. Manev,et al.  Intra-abdominal injection of double-stranded RNA into anesthetized adult Drosophila triggers RNA interference in the central nervous system , 2001, Molecular Psychiatry.

[14]  A. Fire,et al.  Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans , 1998, Nature.

[15]  T. Gorojankina,et al.  Cloning and characterization of a gut‐specific cathepsin L from the aphid Aphis gossypii , 2004, Insect molecular biology.

[16]  X. Belles,et al.  RNAi studies reveal a conserved role for RXR in molting in the cockroach Blattella germanica. , 2006, Journal of insect physiology.

[17]  T. Köcher,et al.  Reverse genetics in the mosquito Anopheles gambiae: targeted disruption of the Defensin gene , 2002, EMBO reports.

[18]  Martin Klingler,et al.  Parental RNAi in Tribolium (Coleoptera) , 2002, Current Biology.

[19]  D. Wagner,et al.  SPLAYED, a Novel SWI/SNF ATPase Homolog, Controls Reproductive Development in Arabidopsis , 2002, Current Biology.

[20]  Yoshiharu Tanaka,et al.  Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster , 2003, Anesthesiology.

[21]  K. Suzuki,et al.  Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). Homology with animal cystatins and transient expression in the ripening process of rice seeds. , 1987, The Journal of biological chemistry.

[22]  D. Dykxhoorn,et al.  Killing the messenger: short RNAs that silence gene expression , 2003, Nature Reviews Molecular Cell Biology.

[23]  P. Abad,et al.  Application of RNA interference to root-knot nematode genes encoding esophageal gland proteins. , 2005, Molecular plant-microbe interactions : MPMI.

[24]  Piero Carninci,et al.  Transcriptome analysis of the aphid bacteriocyte, the symbiotic host cell that harbors an endocellular mutualistic bacterium, Buchnera. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[25]  K. Nakai,et al.  PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. , 1999, Trends in biochemical sciences.

[26]  G. Amdam,et al.  Disruption of vitellogenin gene function in adult honeybees by intra-abdominal injection of double-stranded RNA , 2003, BMC biotechnology.

[27]  Patrick Wincker,et al.  Large-scale gene discovery in the pea aphid Acyrthosiphon pisum (Hemiptera) , 2006, Genome Biology.

[28]  B. Boisson,et al.  Gene silencing in mosquito salivary glands by RNAi , 2006, FEBS letters.

[29]  Pawan Malhotra,et al.  Silencing of Midgut Aminopeptidase N of Spodoptera litura by Double-stranded RNA Establishes Its Role asBacillus thuringiensis Toxin Receptor* , 2002, The Journal of Biological Chemistry.

[30]  M. Friedrich,et al.  Nymphal RNAi: systemic RNAi mediated gene knockdown in juvenile grasshopper , 2005, BMC Biotechnology.

[31]  X. Huang,et al.  CAP3: A DNA sequence assembly program. , 1999, Genome research.