The presence of symbiotic bacteria in flies belonging to the subfamily Tephritinae, which predominantly infest the flower heads of composite flowers (Asteraceae), was investigated. Twenty-five species of flies, collected mainly in northern Italy, were examined. The bacteria adhered to the midgut epithelium in a space external to the peritrophic membrane and therefore not in direct contact with the gut contents. Specific, unique and live, but unculturable bacteria were consistently found in the majority of the fly species and their presence was also shown to be persistent in flies reared under microbiologically controlled conditions and devoid of any residual culturable intestinal bacteria. Sequencing of the small subunit rRNA gene from the novel bacteria indicated that they belonged to the family . Three main strongly supported clades were delineated by phylogenetic trees, the first of which featured a coherent set of sequences displaying gene sequence similarities lower than 96 % compared with recognized taxa. The second and third clades featured cases with higher gene sequence similarities to culturable bacteria, including and , respectively. Relative rate tests were supportive of a fast genetic evolution for the majority of the bacterial symbionts of the subfamily Tephritinae. In agreement with the interpretation suggested in 1929 after pioneering observations made by H. J. Stammer, a symbiotic relationship between the novel bacteria and the tephritid flies is postulated. The origin of this apparently polyphyletic relationship is discussed and a novel candidate organism is proposed for the first clade under the designation ‘ Stammerula tephritidis’.


Article metrics loading...

Loading full text...

Full text loading...



  1. Aksoy, S.(1995). Molecular analysis of the endosymbionts of tsetse flies: 16S rDNA locus and over-expression of a chaperonin. Insect Mol Biol 4, 23–29.[CrossRef] [Google Scholar]
  2. Bandi, C., Damiani, G., Magrassi, L., Grigolo, A., Fani, R. & Sacchi, L.(1994). Flavobacteria as intracellular symbionts in cockroaches. Proc R Soc Lond B Biol Sci 257, 43–48.[CrossRef] [Google Scholar]
  3. Baumann, P. & Moran, N. A.(1997). Non-cultivable microorganisms from symbiotic associations of insects and other hosts. Antonie van Leeuwenhoek 72, 39–48.[CrossRef] [Google Scholar]
  4. Baumann, P., Lai, C., Baumann, L., Rouhbakhsh, D., Moran, N. A. & Clark, M. A.(1995). Mutalistic associations of aphid and prokaryotes: biology of the genus Buchnera. Appl Environ Microbiol 61, 1–7. [Google Scholar]
  5. Baumann, L., Thao, M. L., Hess, J. M., Johnson, M. W. & Baumann, P.(2002). The genetic properties of the primary endosymbionts of mealybugs differ from those of other endosymbionts of plant sap-sucking insects. Appl Environ Microbiol 68, 3198–3205.[CrossRef] [Google Scholar]
  6. Belcari, A.(1985). Presenza di Acanthiophilus helianthi su girasole in Toscana. Inf Fitopatol 35, 23–26. [Google Scholar]
  7. Brynnel, E. U., Kurland, C. G., Moran, N. A. & Andersson, S. G.(1998). Evolutionary rates for tuf genes in endosymbionts of aphids. Mol Biol Evol 15, 574–582.[CrossRef] [Google Scholar]
  8. Buchner, P.(1965).Endosymbiosis of Animals with Plant Microorganisms. New York: Interscience Publishers, Inc.
  9. Campbell, B. C., Bragg, T. S. & Turner, C. E.(1992). Phylogeny of symbiotic bacteria of four weevil species (Coleoptera: Curculionidae) based on analysis of 16S ribosomal DNA. Insect Biochem Mol Biol 22, 415–421.[CrossRef] [Google Scholar]
  10. Capuzzo, C., Firrao, G., Mazzon, L., Squartini, A. & Girolami, V.(2005).Candidatus Erwinia dacicola’, a coevolved symbiotic bacterium of the olive fly Bactrocera oleae (Gmelin). Int J Syst Evol Microbiol 55, 1641–1647.[CrossRef] [Google Scholar]
  11. Clark, M. A., Baumann, L., Munson, M. A., Baumann, P., Campbell, B. C., Duffus, J. E., Osborne, L. S. & Moran, N. A.(1992). The eubacterial endosymbionts of whiteflies (Homoptera: Aleyrodoidea) constitute a lineage distinct from the endosymbionts of aphids and mealybugs. Curr Microbiol 25, 119–123.[CrossRef] [Google Scholar]
  12. Foote, R. H., Blanc, F. L. & Norrbom, A. L.(1993).Handbook of the Fruit Flies (Diptera: Tephritidae) of America North of Mexico. Ithaca, NY: Comstock.
  13. Girolami, V.(1973). Reperti morfo-istologici sulle batteriosimbiosi del Dacus oleae Gmelin e di altri ditteri tripetidi, in natura e negli allevamenti su substrati artificiali. Redia 54, 269–294 (in Italian). [Google Scholar]
  14. Girolami, V.(19783). Fruit fly symbiosis and adult survival: general aspects. In Fruit Flies of Economic Importance, pp. 74–76. Edited by R. Cavalloro, Rotterdam. Proc CEC/IOBC Int Symp, Athens November 1982.
  15. Guindon, S. & Gascuel, O.(2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52, 696–704.[CrossRef] [Google Scholar]
  16. Han, H. Y., Ro, K. E. & McPheron, B. A.(2006). Molecular phylogeny of the subfamily tephritinae (Diptera: Tephritidae) based on mitochondrial 16S rDNA sequences. Mol Cells 22, 78–88. [Google Scholar]
  17. Headrick, D. H. & Goeden, R. D.(1998). The biology of nonfrugivorous tephritid fruit flies. Annu Rev Entomol 43, 217–241.[CrossRef] [Google Scholar]
  18. Heddi, A., Charles, H., Khatchadourian, C., Bonnot, G. & Nardon, P.(1998). Molecular characterization of the principal symbiotic bacteria of the weevil Sitophilus oryzae: a peculiar G+C content of an endocytobiotic DNA. J Mol Evol 47, 52–61.[CrossRef] [Google Scholar]
  19. Hendel, F.(1927). 49. Trypetidae. In Die Fliegen der Palaearktischen Region. Edited by E. Lindner. Stuttgart, Germany: Schweitzerbart'sche Verlagsbuchhandlung.
  20. Huelsenbeck, J. P. & Ronquist, F.(2001). MrBayes: Bayesian inference of phylogeny. Bioinformatics 17, 754–755.[CrossRef] [Google Scholar]
  21. Kumar, S.(1996). Phyltest: Phylogenetic Hypothesis Testing Software, version 2.0. University Park, PA: Penn State University.
  22. Kumar, S., Tamura, K. & Nei, M.(2004).mega3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.[CrossRef] [Google Scholar]
  23. Lefèvre, C., Charles, H., Vallier, A., Delobel, B., Farrell, B. & Heddi, A.(2004). Endosymbiont phylogenesis in the Dryophthoridae weevils: evidence for bacterial replacement. Mol Biol Evol 21, 965–973.[CrossRef] [Google Scholar]
  24. Lloyd, A. C., Drew, R. A. I., Teakle, D. S. & Hayward, A. C.(1986). Bacteria associated with some Dacus species (Diptera: Tephritidae) and their host fruit in Queensland. Aust J Biol Sci 39, 361–368. [Google Scholar]
  25. Merz, B.(1994).Diptera: Tephritidae. Insecta Helvetica Fauna, vol. 10. Lausanne: Musée d'Histoire Naturelle de Neuchatel.
  26. Moran, N. A.(1996). Accelerated evolution and Muller's ratchet in endosymbiotic bacteria. Proc Natl Acad Sci U S A 93, 2873–2878.[CrossRef] [Google Scholar]
  27. Moran, N. A., Russell, J. A., Koga, R. & Fukatsu, T.(2005). Evolutionary relationships of three new species of enterobacteriaceae living as symbionts of aphids and other insects. Appl Environ Microbiol 71, 3302–3310.[CrossRef] [Google Scholar]
  28. Munson, M. A., Baumann, P. & Moran, N. A.(1992). Phylogenetic relationships of endosymbionts of mealybugs (Homoptera: Pseudococcidae) based on 16S rDNA sequences. Mol Phylogenet Evol 1, 26–30.[CrossRef] [Google Scholar]
  29. Muse, S. V. & Weir, B. S.(1992). Testing for equality of evolutionary rates. Genetics 132, 269–276. [Google Scholar]
  30. Noda, H., Nakashima, N. & Koizumi, M.(1995). Phylogenetic position of yeast-like symbiotes of rice planthoppers based on partial 18S rDNA sequences. Insect Biochem Mol Biol 25, 639–646.[CrossRef] [Google Scholar]
  31. Nylander, J. A. A.(2004). MrModeltest v2.1 [computer program]. http://www.ebc.uu.se/systzoo/staff/nylander.html
  32. Palmano, S., Firrao, G. & Locci, R.(2000). Sequence analysis of domains III and IV of the 23S rRNA gene of verticillate streptomycetes. Int J Syst Evol Microbiol 50, 1187–1191.[CrossRef] [Google Scholar]
  33. Petri, L.(1909).Ricerche Sopra i Batteri Intestinali della Mosca Olearia. Roma: Memorie della Regia Stazione di Patologia Vegetale di Roma.
  34. Pignatti, S.(1982).Flora d'Italia. Edagricole, Bologna.
  35. Posada, D. & Buckley, T. R.(2004). Model selection and model averaging in phylogenetics: advantages of AIC and Bayesian approaches over likelihood ratio tests. Syst Biol 53, 793–808.[CrossRef] [Google Scholar]
  36. Posada, D. & Crandall, K. A.(1998).modeltest: testing the model of DNA substitution. Bioinformatics 14, 817–818.[CrossRef] [Google Scholar]
  37. Ricci, C. & Ciriciofolo, E.(1983). Osservazioni sull'Acanthiophilus helianthi Rossi (Diptera Tephritidae) dannoso al cartamo in Italia centrale. Redia 66, 577–592. [Google Scholar]
  38. Rossiter, M. C., Howard, D. J. & Bush, G. L.(1983). Symbiotic bacteria of Rhagoletis pomonella, pp. 77–82. Edited by R. Cavalloro, Rotterdam. Proc CEC/IOBC Int Symp, Athens November 1982.
  39. Rouhbakhsh, D., Lai, C.-Y., von Dohlen, C. D., Baumann, L., Baumann, P., Moran, N. A. & Voegtlin, D. J.(1996). The tryptophan biosynthetic pathway of aphid endosymbionts (Buchnera): genetics and evolution of plasmid-associated trpEG within the Aphididae. J Mol Evol 42, 414–421.[CrossRef] [Google Scholar]
  40. Schröder, D., Deppisch, H., Obermayer, M., Krohne, G., Stackebrandt, E., Hölldobler, B., Goebel, W. & Gross, R.(1996). Intracellular endosymbiotic bacteria of Camponotus species (carpenter ants): systematics, evolution and ultrastructural analysis. Mol Microbiol 21, 479–489.[CrossRef] [Google Scholar]
  41. Séguy, E.(1934). Diptères (Brachycères) (Muscidae Acalypterae et Scatophagidae), vol. 28. Paris: Faune de France.
  42. Stammer, H. J.(1929). Die bakteriensymbiose der trypetiden (Diptera). Zoomorphology 15, 481–523. [Google Scholar]
  43. Swofford, D. L.(2002).paup*: Phylogenetic analysis using parsimony (and other methods), version 4. Sunderland, MA: Sinauer Associates.
  44. Weisburg, W. G., Barns, S. M., Pelletier, D. E. A. & Lane, D. J.(1991). 16S Ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697–703. [Google Scholar]
  45. Wernegreen, J. J.(2002). Genome evolution in bacterial endosymbionts of insects. Nat Rev Genet 3, 850–861.[CrossRef] [Google Scholar]
  46. White, I. M.(1988).Tephritid Flies Diptera: Tephritidae. Handbook for the Identification of British Insects, vol. 10, Part 5a. London: Royal Entomological Society of London.
  47. Wu, C. I. & Li, W. H.(1985). Evidence for higher rates of nucleotide substitution in rodents than in man. Proc Natl Acad Sci U S A 82, 1741–1745.[CrossRef] [Google Scholar]
  48. Zwölfer, H.(1983). Life systems and strategies of resource exploitation in tephritids. In Fruit Flies of Economic Importance, pp. 16–30. Edited by R. Cavalloro. Rotterdam. Proc CEC/IOBC Int Symp, Athens November 1982.

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error