Molecular Genetics and Genomics

, Volume 269, Issue 6, pp 753–764 | Cite as

A targeted approach to the identification of candidate genes determining susceptibility to Plasmodium gallinaceum in Aedes aegypti

  • I. Morlais
  • A. Mori
  • J. R. Schneider
  • D. W. Severson
Original Paper


The malaria parasite, Plasmodium , has evolved an intricate life cycle that includes stages specific to a mosquito vector and to the vertebrate host. The mosquito midgut represents the first barrier Plasmodium parasites encounter following their ingestion with a blood meal from an infected vertebrate. Elucidation of the molecular interaction between the parasite and the mosquito could help identify novel approaches to preventing parasite development and subsequent transmission to vertebrates. We have used an integrated Bulked Segregant Analysis-Differential Display (BSA-DD) approach to target genes expressed that are in the midgut and located within two genome regions involved in determining susceptibility to P. gallinaceum in the mosquito Aedes aegypti. A total of twenty-two genes were identified and characterized, including five genes with no homologues in public sequence databases. Eight of these genes were mapped genetically to intervals on chromosome 2 that contain two quantitative trait loci (QTLs) that determine susceptibility to infection by P. gallinaceum. Expression analysis revealed several expression patterns, and ten genes were specifically or preferentially expressed in the midgut of adult females. Real-time PCR quantification of expression with respect to the time of blood meal ingestion and infection status in mosquito strains permissive and refractory for malaria revealed a differential expression pattern for seven genes. These represent candidate genes that may influence the ability of the mosquito vector to support the development of Plasmodium parasites. Here we describe their isolation and discuss their putative roles in parasite-mosquito interactions and their use as potential targets in strategies designed to block transmission of malaria.


Bulked segregant analysis Differential display Malaria Quantitative trait loci (QTLs) Vector competence 


  1. Akey JM, Sosnoski D, Parra E, Dios S, Hiester K, Su B, Bonilla C, Jin L, Shriver MD (2001) Melting curve analysis of SNPs (McSNP): a gel-free and inexpensive approach for SNP genotyping. Biotechniques 30:358–367PubMedGoogle Scholar
  2. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedGoogle Scholar
  3. Arca B, Lombardo F, de Lara Capurro M, della Torre A, Dimopoulos G, James AA, Coluzzi M (1999) Trapping cDNAs encoding secreted proteins from the salivary glands of the malaria vector Anopheles gambiae. Proc Natl Acad Sci USA 96:1516–1521CrossRefPubMedGoogle Scholar
  4. Bertioli DJ, Schlichter UHA, Adams MJ, Burrows PK, Steinbiss HH, Antoniw JF (1995) An analysis of differential display shows a strong bias towards high copy number messenger RNAs. Nucleic Acids Res 23:4520–4523PubMedGoogle Scholar
  5. Catteruccia F, Nolan T, Blass C, Muller HM, Crisanti A, Kafatos FC, Loukeris TG (2000) Toward Anopheles transformation: Minos element activity in anopheline cells and embryos. Proc Natl Acad Sci USA 97:2157–2162CrossRefPubMedGoogle Scholar
  6. Coates CJ (2000) Malaria. A mosquito transformed. Nature 405:900–901CrossRefPubMedGoogle Scholar
  7. Crabb JW, Carlson A, Chen Y, Goldflam S, Intres R, West KA, Hulmes JD, Kapron JT, Luck LA, Horwitz J, Bok D (1998) Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein. Protein Sci 7:746–757PubMedGoogle Scholar
  8. Delmas B, Gelfi J, L'Haridon R, Vogel LK, Sjostrom H, Noren O, Laude H (1992) Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV. Nature 357:417–420CrossRefPubMedGoogle Scholar
  9. Dessens JT, Beetsma AL, Dimopoulos G, Wengelnik K, Crisanti A, Kafatos FC, Sinden RE (1999) CTRP is essential for mosquito infection by malaria ookinetes. EMBO J 18:6221–6227CrossRefPubMedGoogle Scholar
  10. Dimopoulos G, Richman A, della Torre A, Kafatos FC, Louis C (1996) Identification and characterization of differentially expressed cDNAs of the vector mosquito, Anopheles gambiae. Proc Natl Acad Sci USA 93:13066–13071PubMedGoogle Scholar
  11. Dimopoulos G, Seeley D, Wolf A, Kafatos FC (1998) Malaria infection of the mosquito Anopheles gambiae activates immune- responsive genes during critical transition stages of the parasite life cycle. EMBO J 17:6115–6123CrossRefPubMedGoogle Scholar
  12. Dimopoulos G, Muller HM, Levashina EA, Kafatos FC (2001) Innate immune defense against malaria infection in the mosquito. Curr Opin Immunol 13:79–88PubMedGoogle Scholar
  13. Dunkov BC, Zhang D, Choumarov K, Winzerling JJ, Law JH (1995) Isolation and characterization of mosquito ferritin and cloning of a cDNA that encodes one subunit. Arch Insect Biochem Physiol 29:293–307PubMedGoogle Scholar
  14. Feldmann AM, Billingsley PF, Savelkoul E (1990) Bloodmeal digestion by strains of Anopheles stephensi Liston (Diptera: Culicidae) of differing susceptibility to Plasmodium falciparum. Parasitology 101:193–200PubMedGoogle Scholar
  15. Feyereisen R (1999) Insect P450 enzymes. Annu Rev Entomol 44:507–533PubMedGoogle Scholar
  16. Ghosh A, Edwards MJ, Jacobs-Lorena M (2000) The journey of the malaria parasite in the mosquito: hopes for the new century. Parasitol Today 16:196–201CrossRefPubMedGoogle Scholar
  17. Giugni TD, Soderberg C, Ham DJ, Bautista RM, Hedlund KO, Moller E, Zaia JA (1996) Neutralization of human cytomegalovirus by human CD13-specific antibodies. J Infect Dis 173:1062–1071PubMedGoogle Scholar
  18. Hoffmann JA (1995) Innate immunity of insects. Curr Opin Immunol 7:4–10CrossRefPubMedGoogle Scholar
  19. Huber M, Cabib E, Miller LH (1991) Malaria parasite chitinase and penetration of the mosquito peritrophic membrane. Proc Natl Acad Sci USA 88:2807–2810PubMedGoogle Scholar
  20. Hultmark D (1993) Immune reactions in Drosophila and other insects: a model for innate immunity. Trends Genet 9:178–83.PubMedGoogle Scholar
  21. Jasinskiene N, Coates CJ, Benedict MQ, Cornel AJ, Rafferty CS, James AA, Collins FH (1998) Stable transformation of the yellow fever mosquito, Aedes aegypti , with the Hermes element from the housefly. Proc Natl Acad Sci USA 95:3743–3747PubMedGoogle Scholar
  22. Jiang Q, Hall M, Noriega FG, Wells M (1997) cDNA cloning and pattern of expression of an adult, female-specific chymotrypsin from Aedes aegypti midgut. Insect Biochem Mol Biol 27:283–289CrossRefPubMedGoogle Scholar
  23. Kokoza V, Ahmed A, Cho WL, Jasinskiene N, James AA, Raikhel A (2000) Engineering blood meal-activated systemic immunity in the yellow fever mosquito, Aedes aegypti. Proc Natl Acad Sci USA 97:9144–9149CrossRefPubMedGoogle Scholar
  24. Kosambi DD (1944) The estimation of map distance from recombination values. Ann Eugen 12:172–175Google Scholar
  25. Lander ES, Green P, Abrahamson J, Barlow A, Daly MJ, Lincoln SE, Newburg L (1987) MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1:174–181PubMedGoogle Scholar
  26. Ledakis P, Tanimura H, Fojo T (1998) Limitations of differential display. Biochim Biophys Res Comm 251:653–656CrossRefGoogle Scholar
  27. Liang P, Pardee AB (1992) Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967–971PubMedGoogle Scholar
  28. Lincoln SE, Lander ES (1990) Mapping genes controlling quantitative traits using MAPMAKER/QTL. Whitehead Institute for Biomedical Research, Cambridge, Mass.Google Scholar
  29. Luckhart S, Vodovotz Y, Cui L, Rosenberg R (1998) The mosquito Anopheles stephensi limits malaria parasite development with inducible synthesis of nitric oxide. Proc Natl Acad Sci USA 95:5700–5705CrossRefPubMedGoogle Scholar
  30. Moreira LA, Edwards MJ, Adhami F, Jasinskiene N, James AA, Jacobs-Lorena M (2000) Robust gut-specific gene expression in transgenic Aedes aegypti mosquitoes. Proc Natl Acad Sci USA 97:10895–10898CrossRefPubMedGoogle Scholar
  31. Morlais I, Severson DW (2001) Identification of a polymorphic mucin-like gene expressed in the midgut of the mosquito, Aedes aegypti , using an integrated bulked segregant and differential display analysis. Genetics 158:1125–1136PubMedGoogle Scholar
  32. Muller HM, Catteruccia F, Vizioli J, della Torre A, Crisanti A (1995) Constitutive and blood meal-induced trypsin genes in Anopheles gambiae. Exp Parasitol 81:371–385PubMedGoogle Scholar
  33. Nakanishi K, Yaoi K, Nagino Y, Hara H, Kitami M, Atsumi S, Miura N, Sato R (2002) Aminopeptidase N isoforms from the midgut of Bombyx mori and Plutella xylostella —their classification and the factors that detrmine their binding specificity to Bacillus thuringiensis CryIA toxin. FEBS Lett 519:215–220CrossRefPubMedGoogle Scholar
  34. Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6Google Scholar
  35. Oduol F, Xu J, Niare O, Natarajan R, Vernick KD (2000) Genes identified by an expression screen of the vector mosquito Anopheles gambiae display differential molecular immune response to malaria parasites and bacteria. Proc Natl Acad Sci USA 97:11397–11402CrossRefPubMedGoogle Scholar
  36. Oppermann UC, Filling C, Jornvall H (2001) Forms and functions of human SDR enzymes. Chem Biol Interact 130–132:699–705Google Scholar
  37. Pautot V, Holzer FM, Reisch B, Walling LL (1993) Leucine aminopeptidase—an inducible component of the defense response in Lycopersicon esculentum (tomato). Proc Natl Acad Sci USA 90:9906–9910PubMedGoogle Scholar
  38. Pomes A, Melen E, Vailes LD, Retief JD, Arruda LK, Chapman MD (1998) Novel allergen structures with tandem amino acid repeats derived from German and American cockroach. J Biol Chem 273:30801–30807CrossRefPubMedGoogle Scholar
  39. Richards AG, Richards PA (1977) The peritrophic membranes of insects. Annu Rev Entomol 22:219–240CrossRefPubMedGoogle Scholar
  40. Richman AM, Dimopoulos G, Seeley D, Kafatos FC (1997) Plasmodium activates the innate immune response of Anopheles gambiae mosquitoes. EMBO J 16:6114–6119CrossRefPubMedGoogle Scholar
  41. Rose RL, Goh D, Thompson DM, Verma KD, Heckel DG, Gahan LJ, Roe RM, Hodgson E (1997) Cytochrome P450 (CYP)9A1 in Heliothis virescens: the first member of a new CYP family. Insect Biochem Mol Biol 27:605–615CrossRefPubMedGoogle Scholar
  42. Rosenfeld A, Vanderberg JP (1999) Plasmodium berghei: induction of aminopeptidase in malaria-resistant strain of Anopheles gambiae. Exp Parasitol 93:101–104CrossRefPubMedGoogle Scholar
  43. Schmid KJ, Tautz D (1997) A screen for fast evolving genes from Drosophila. Proc Natl Acad Sci USA 94:9746–9750CrossRefPubMedGoogle Scholar
  44. Scott JG, Liu N, Wen Z (1998) Insect cytochromes P450: diversity, insecticide resistance and tolerance to plant toxins. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 121:147–155CrossRefPubMedGoogle Scholar
  45. Severson DW (1997) RFLP analysis of insect genomes. In: Crampton JM, Beard CB, Louis C (eds) The Molecular biology of insect disease vectors. Chapman and Hall, London, pp 309–320Google Scholar
  46. Severson DW, Thathy V, Mori A, Zhang Y, Christensen BM (1995) Restriction fragment length polymorphism mapping of quantitative trait loci for malaria parasite susceptibility in the mosquito Aedes aegypti. Genetics 139:1711–1717PubMedGoogle Scholar
  47. Severson DW, Meece JK, Lovin DD, Saha G, Morlais I (2002) Linkage map organization of expressed sequence tags and sequence tagged sites in the mosquito, Aedes aegypti. Insect Mol Biol 11:371–378CrossRefPubMedGoogle Scholar
  48. Shahabuddin M, Criscio M, Kaslow DC (1995) Unique specificity of in vitro inhibition of mosquito midgut trypsin-like activity correlates with in vivo inhibition of malaria parasite infectivity. Exp Parasitol 80:212–219CrossRefPubMedGoogle Scholar
  49. Shao Z, Cui Y, Liu X, Yi H, Ji J, Yu Z (1998) Processing of delta-endotoxin of Bacillus thuringiensis subsp. kurstaki HD-1 in Heliothis armigera midgut juice and the effects of protease inhibitors. J Invertebr Pathol 72:73–81CrossRefPubMedGoogle Scholar
  50. Shen Z, Dimopoulos G, Kafatos FC, Jacobs-Lorena M (1999) A cell surface mucin specifically expressed in the midgut of the malaria mosquito Anopheles gambiae. Proc Natl Acad Sci USA 96:5610–5615CrossRefPubMedGoogle Scholar
  51. Shen Z, Edwards MJ, Jacobs-Lorena M (2000) A gut-specific serine protease from the malaria vector Anopheles gambiae is downregulated after blood ingestion. Insect Mol Biol 9:223–229CrossRefPubMedGoogle Scholar
  52. Sieber KP, Huber M, Kaslow D, Banks SM, Torii M, Aikawa M, Miller LH (1991) The peritrophic membrane as a barrier: its penetration by Plasmodium gallinaceum and the effect of a monoclonal antibody to ookinetes. Exp Parasitol 72:145–156PubMedGoogle Scholar
  53. Stevens JL, Snyder MJ, Koener JF, Feyereisen R (2000) Inducible P450s of the CYP9 family from larval Manduca sexta midgut. Insect Biochem Mol Biol 30:559–568CrossRefPubMedGoogle Scholar
  54. Tautz D, Schmid KJ (1998) From genes to individuals: developmental genes and the generation of the phenotype. Philos Trans R Soc Lond B Biol Sci 353:231–240CrossRefPubMedGoogle Scholar
  55. Tellam RL, Wijffels G, Willadsen P (1999) Peritrophic matrix proteins. Insect Biochem Mol Biol 29:87–101CrossRefPubMedGoogle Scholar
  56. Thathy V, Severson DW, Christensen BM (1994) Reinterpretation of the genetics of susceptibility of Aedes aegypti to Plasmodium gallinaceum. J Parasitol 80:705–712PubMedGoogle Scholar
  57. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680PubMedGoogle Scholar
  58. Vinetz JM, Valenzuela JG, Specht CA, Aravind L, Langer RC, Ribeiro JM, Kaslow DC (2000) Chitinases of the avian malaria parasite Plasmodium gallinaceum, a class of enzymes necessary for parasite invasion of the mosquito midgut. J Biol Chem 275:10331–10341CrossRefPubMedGoogle Scholar
  59. Wu CH, Wang NM, Lee MF, Kao CY, Luo SF (1998) Cloning of the American cockroach Cr-PII allergens: evidence for the existence of cross-reactive allergens between species. J Allergy Clin Immunol 101:832–840PubMedGoogle Scholar
  60. Zieler H, Nawrocki JP, Shahabuddin M (1999) Plasmodium gallinaceum ookinetes adhere specifically to the midgut epithelium of Aedes aegypti by interaction with a carbohydrate ligand. J Exp Biol 202:485–495PubMedGoogle Scholar
  61. Zimmer S, Stocker A, Sarbolouki MN, Spycher SE, Sassoon J, Azzi A (2000) A novel human tocopherol-associated protein: cloning, in vitro expression, and characterization. J Biol Chem 275:25672–25680CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • I. Morlais
    • 1
  • A. Mori
    • 1
  • J. R. Schneider
    • 1
  • D. W. Severson
    • 1
  1. 1.Center for Tropical Disease Research and Training, Department of Biological SciencesUniversity of Notre DameNotre DameUSA

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