Microbial Ecology

, Volume 64, Issue 1, pp 268–278 | Cite as

Gut-Associated Bacteria Throughout the Life Cycle of the Bark Beetle Dendroctonus rhizophagus Thomas and Bright (Curculionidae: Scolytinae) and Their Cellulolytic Activities

  • Jesús Morales-Jiménez
  • Gerardo Zúñiga
  • Hugo C. Ramírez-Saad
  • César Hernández-RodríguezEmail author


Dendroctonus rhizophagus Thomas and Bright (Curculionidae: Scolytinae) is an endemic economically important insect of the Sierra Madre Occidental in Mexico. This bark beetle has an atypical behavior within the genus because just one beetle couple colonizes and kills seedlings and young trees of 11 pine species. In this work, the bacteria associated with the Dendroctonus rhizophagus gut were analyzed by culture-dependent and culture-independent methods. Analysis of 16S rRNA sequences amplified directly from isolates of gut bacteria suggests that the bacterial community associated with Dendroctonus rhizophagus, like that of other Dendroctonus spp. and Ips pini, is limited in number. Nine bacterial genera of γ-Proteobacteria and Actinobacteria classes were detected in the gut of Dendroctonus rhizophagus. Stenotrophomonas and Rahnella genera were the most frequently found bacteria from Dendroctonus rhizophagus gut throughout their life cycle. Stenotrophomonas maltophilia, Ponticoccus gilvus, and Kocuria marina showed cellulolytic activity in vitro. Stenotrophomonas maltophilia, Rahnella aquatilis, Raoultella terrigena, Ponticoccus gilvus, and Kocuria marina associated with larvae or adults of Dendroctonus rhizophagus could be implicated in nitrogen fixation and cellulose breakdown, important roles associated to insect development and fitness, especially under the particularly difficult life conditions of this beetle.


Bacterial Community Bark Beetle Pseudomonas Fluorescens Stenotrophomonas Maltophilia Cellulolytic Activity 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We would like to thank Félix Aguirre Garrido for the technical assistance with DGGE. This work was supported by grants SIP 20080688, 20090738, 20100430, and 20111068; IPN; and CONAFOR-CONACyT 69539. Jesús Morales-Jiménez would like to thank CONACyT, and PIFI-IPN for the scholarships.


  1. 1.
    Adams AS, Adams SM, Currie CR, Gillette NE, Raffa KF (2010) Geographic variation in bacterial communities associated with the red turpentine beetle (Coleoptera: Curculionidae). Env Entomol 39:406–414CrossRefGoogle Scholar
  2. 2.
    Adams AS, Boone CK, Bohlmann J, Raffa KF (2011) Responses of bark beetle-associated bacteria to host monoterpenes and their relationship to insect life histories. J Chem Ecol 37:808–817PubMedCrossRefGoogle Scholar
  3. 3.
    Altschul SF, Madden TL, Schäffer 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 25:3389–3402PubMedCrossRefGoogle Scholar
  4. 4.
    Behar A, Yuval B, Jurkevitch E (2005) Enterobacteria mediated nitrogen fixation in natural populations of the fruit fly Ceratitis capitata. Mol Ecol 14:2637–2643PubMedCrossRefGoogle Scholar
  5. 5.
    Bicas JL, Fontanille P, Pastore GM, Larroche C (2008) Characterization of monoterpene biotransformation in two pseudomonads. J Appl Microbiol 105:1991–2001PubMedCrossRefGoogle Scholar
  6. 6.
    Brand JM, Bracke JW, Markovetz AJ, Wood DL, Browne AJ (1975) Production of verbenol pheromone by a bacterium isolated from bark beetles. Nature 254:136–137PubMedCrossRefGoogle Scholar
  7. 7.
    Breznak JA (1982) Intestinal microbiota of termites and other xylophagous insects. Annu Rev Microbiol 36:323–343PubMedCrossRefGoogle Scholar
  8. 8.
    Brune A, Friedrich M (2000) Microecology of the termite gut: structure and function on a microscale. J Curr Opin Microbiol 3:263–269CrossRefGoogle Scholar
  9. 9.
    Campanella JJ, Bitincka L, Smalley J (2003) MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. BMC Bioinforma 4:29CrossRefGoogle Scholar
  10. 10.
    Cankar K, Kraigher H, Ravnikar M, Rupnik M (2005) Bacterial endophytes from seeds of Norway spruce (Picea abies L. Karst). Microbiol Lett 244:341–345CrossRefGoogle Scholar
  11. 11.
    Carpenter KJ, Horak A, Keeling PJ (2010) Phylogenetic position and morphology of Spirotrichosomidae (Parabasalia): new evidence from Leptospironympha of Cryptocercus punctulatus. Protist 161:122–132PubMedCrossRefGoogle Scholar
  12. 12.
    Cibrián-Tovar D, Méndez-Montiel JT, Campos-Bolaños R, Yates HO III, Flores-Lara J (1995) Insectos forestales de México [Forest Insects of Mexico]. Universidad Autónoma Chapingo, ChapingoGoogle Scholar
  13. 13.
    Cruden DL, Markovetz AJ (1987) Microbial ecology of the cockroach gut. Annu Rev Microbiol 41:617–643PubMedCrossRefGoogle Scholar
  14. 14.
    Cunningham AA, Frank JM, Croft P, Clarke D, Pearce-Kelly P (1997) Mortality of captive British wartbiter crickets: implications for reintroduction programs. J Wild Dis 33:673–676Google Scholar
  15. 15.
    Delalibera I, Handelsman J, Raffa KF (2005) Contrasts in cellulolytic activities of gut microorganisms between the wood borer, Saperda vestita (Coleoptera: Cerambycidae), and the bark beetles, Ips pini and Dendroctonus frontalis (Coleoptera: Curculionidae). Env Entomol 34:541–547CrossRefGoogle Scholar
  16. 16.
    Delalibera I, Vasanthakumar A, Burwitz BJ, Schloss PD, Klepzig KD, Handelsman J, Raffa KF (2007) Composition of the bacterial community in the gut of the pine engraver, Ips pini (Say) (Coleoptera) colonizing red pine. Symbiosis 43:97–104Google Scholar
  17. 17.
    Douglas AE (2009) The microbial dimension in insect nutritional ecology. Funct Ecol 23:38–47CrossRefGoogle Scholar
  18. 18.
    Dutkiewicz J, Krysińska-Traczyk E, Prazmo Z, Skoŕska C, Sitkowska J (2001) Exposure to airborne microorganisms in Polish sawmills. Ann Agric Env Med 8:71–80Google Scholar
  19. 19.
    Estrada-Murrieta O (1983) Biología del descortezador del renuevo de pino Dendroctonus rhizophagus T. y B. (Col.: Scolytidae) en la región Mesa del Huracán, Chih. Bachelor Thesis. Universidad Autónoma Chapingo, MéxicoGoogle Scholar
  20. 20.
    Genta FA, Dillon RJ, Terra WR, Ferreira C (2006) Potential role for gut microbiota in cell wall digestion and glucoside detoxification in Tenebrio molitor larvae. J Insect Physiol 52:593–601PubMedCrossRefGoogle Scholar
  21. 21.
    Guindon S, Gascuel O (2003) PhyML—a simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704PubMedCrossRefGoogle Scholar
  22. 22.
    Grünwald S, Pilhofer M, Höll W (2010) Microbial associations in gut systems of wood- and bark-inhabiting longhorned beetles [Coleoptera: Cerambycidae]. Syst Appl Microbiol 33:25–34PubMedCrossRefGoogle Scholar
  23. 23.
    Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98Google Scholar
  24. 24.
    Hansen DS, Aucken HM, Abiola T, Podschun R (2004) Recommended test panel for differentiation of Klebsiella species on the basis of a trilateral interlaboratory evaluation of 18 biochemical tests. J Clin Microbiol 42:3665–3669PubMedCrossRefGoogle Scholar
  25. 25.
    Hoffman CS, Winston F (1987) A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57:267–272PubMedCrossRefGoogle Scholar
  26. 26.
    Hongoh Y, Ohkuma M, Kudo T (2003) Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera; Rhinotermitidae). FEMS Microbiol Ecol 44:231–242PubMedCrossRefGoogle Scholar
  27. 27.
    Hongoh Y (2011) Toward the functional analysis of uncultivable, symbiotic microorganisms in the termite gut. Cell Mol Life Sci 68:1311–1325PubMedCrossRefGoogle Scholar
  28. 28.
    Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon; a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317–2319PubMedCrossRefGoogle Scholar
  29. 29.
    Izumi H, Anderson IC, Alexander IJ, Killham K, Moore ERB (2006) Endobacteria in some ectomycorrhiza of Scots pine (Pinus sylvestris). FEMS Microbiol Ecol 56:34–43PubMedCrossRefGoogle Scholar
  30. 30.
    Janson EM, Stireman JO, Singer MS, Abbot P (2008) Phytophagous insect-microbe mutualisms and adaptive evolutionary diversification. Evolution 62:997–1012PubMedCrossRefGoogle Scholar
  31. 31.
    Jones KG (1981) Bald cypress allelochemicals and the inhibition of silkworm enteric microorganisms: some ecological considerations. J Chem Ecol 16:1385–1397Google Scholar
  32. 32.
    Klepzig KD, Adams AS, Handelsman J, Raffa KF (2009) Symbioses: a key driver of insect physiological processes, ecological interactions, evolutionary diversification, and impacts on humans. Env Entomol 38:67–77CrossRefGoogle Scholar
  33. 33.
    Kuhnigk T, König H (1997) Degradation of dimeric lignin model compounds by aerobic bacteria isolated from the hindgut of xylophagous termites. J Basic Microbiol 37:205–211PubMedCrossRefGoogle Scholar
  34. 34.
    Mendoza MG, Salinas-Moreno Y, Olivo-Martínez A, Zúniga G (2011) Factors influencing the geographical distribution of Dendroctonus rhizophagus (Coleoptera: Curculionidae: Scolytinae) in the Sierra Madre Occidental, México. Env Entomol 40:549–559CrossRefGoogle Scholar
  35. 35.
    Morales-Jiménez J, Zúñiga G, Villa-Tanaca L, Hernández-Rodríguez C (2009) Bacterial community and nitrogen fixation in the red turpentine beetle, Dendroctonus valens LeConte (Coleoptera: Curculionidae: Scolytinae). Microb Ecol 58:879–891PubMedCrossRefGoogle Scholar
  36. 36.
    Moran NA, Tran P, Gerardo NM (2005) Symbiosis and insect diversification: an ancient symbiont of sap-feeding insects from the bacterial phylum Bacteroidetes. Appl Environ Microbiol 12:8802–8810CrossRefGoogle Scholar
  37. 37.
    Muyzer G, De Waall EC, Vitterlinden AG (1993) Profiling of complete microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rDNA. Appl Environ Microbiol 59:695–700PubMedGoogle Scholar
  38. 38.
    Nakabachi A, Ishikawa H (1999) Provision of riboflavin to the host aphid, Acyrthosiphon pisum, by endosymbiotic bacteria, Buchnera. J Insect Physiol 45:1–6PubMedCrossRefGoogle Scholar
  39. 39.
    Nakajima H, Hongoh Y, Usami R, Kudo T, Ohkuma M (2005) Spatial distributions of bacterial phylotypes in the gut of the termite Reticulitermes speratus and the bacterial community colonizing the gut epithelium. FEMS Microbiol Ecol 54:247–255PubMedCrossRefGoogle Scholar
  40. 40.
    Pinto-Tomás AA, Anderson MA, Suen G, Stevenson DM, Chu FS, Cleland WW, Weimer PJ, Currie CR (2009) Symbiotic nitrogen fixation in the fungus gardens of leaf-cutter ants. Science 326:1120–1123PubMedCrossRefGoogle Scholar
  41. 41.
    Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14:817–818PubMedCrossRefGoogle Scholar
  42. 42.
    Posada D, Buckley TR (2004) Model selection and model averaging in phylogenetics: advantages of Akaike information criterion a Bayesian approaches over likelihood ratio tests. Syst Biol 53:793–808PubMedCrossRefGoogle Scholar
  43. 43.
    Potrikus CJ, Breznak JA (1981) Gut bacteria recycle uric-acid nitrogen in termites—a strategy for nutrient conservation. Proc Natl Acad Sci USA 78:4601–4605PubMedCrossRefGoogle Scholar
  44. 44.
    Relman DA (1993) Universal bacterial 16S rRNA amplification and sequencing. American Society of Microbiology. Washington, DC, pp 489–495Google Scholar
  45. 45.
    Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New YorkGoogle Scholar
  46. 46.
    Sanguinetti CJ, Dias-Neto E, Simpson AJ (1994) Rapid silver staining and recovery of PCR products separated on polyacrylamide gels. Biotech 17:915–919Google Scholar
  47. 47.
    Schloss PD, Handelsman J (2005) Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbiol 71:1501–1506PubMedCrossRefGoogle Scholar
  48. 48.
    Schloss PD, Delalibera I, Handelsman J, Raffa KF (2006) Bacteria associated with the guts of two wood boring beetles: Anoplophora glabripennis and Saperda vestita (Cerambycidae). Env Entomol 35:625–629CrossRefGoogle Scholar
  49. 49.
    Six DL, Klepzig KD (2004) Dendroctonus bark beetles as model systems for studies on symbiosis. Symbiosis 37:207–232Google Scholar
  50. 50.
    Teather RM, Wood PJ (1982) Use of Congo red–polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl Environ Microbiol 43:777–780PubMedGoogle Scholar
  51. 51.
    Thomas JR, Bright DE (1970) A new species of Dendroctonus (Coleoptera: Scolytidae) from Mexico. Can Entomol 102:479–483CrossRefGoogle Scholar
  52. 52.
    Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882PubMedCrossRefGoogle Scholar
  53. 53.
    Trombetta D, Castelli F, Sarpietro MG, Venuti V, Cristani M, Daniele V, Saija A, Mazzanti G, Bisignano G (2005) Mechanisms of antibacterial action of three monoterpenes. Antim Agents Chemoth 49:2474–2478CrossRefGoogle Scholar
  54. 54.
    Vasanthakumar A, Delalibera I, Handelsman J, Klepzig KD, Schloss PD, Raffa KF (2006) Characterization of gut-associated bacteria in larvae and adults of the southern pine beetle, Dendroctonus frontalis Zimmermann. Env Entomol 35:1710–1717CrossRefGoogle Scholar
  55. 55.
    Vasanthakumar A, Handelsman J, Schloss PD, Bauer LS, Raffa KF (2008) Gut microbiota of an invasive subcortical beetle, Agrilus planipennis Fairmaire, across various life stages. Env Entomol 37:1344–1353CrossRefGoogle Scholar
  56. 56.
    Wenzel M, Schonig I, Berchtold M, Kampfer P, Konig H (2002) Aerobic and facultatively anaerobic cellulolytic bacteria from the gut of the termite Zootermopsis angusticollis. J Appl Microbiol 92:32–40PubMedCrossRefGoogle Scholar
  57. 57.
    Williams JGK, Kubelik AR, Livak KJ, Rafalsky JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18:6531–6535PubMedCrossRefGoogle Scholar
  58. 58.
    Wood SL (1982) The bark and ambrosia beetle of North and Central America (Coleoptera: Scolytidae). A taxonomic monograph. Great Basin Nat Mem 6:1–1359Google Scholar
  59. 59.
    Yilmaz H, Sezen K, Kati H, Demirbağ V (2006) The first study on the bacterial flora of the European spruce bark beetle, Dendroctonus micans (Coleoptera: Scolytidae). Biologia 61:679–686CrossRefGoogle Scholar
  60. 60.
    Yu H, Wang Z, Liu L, Xia Y, Cao Y, Yin Y (2008) Analysis of the intestinal microflora in Hepialus gonggaensis larvae using 16S rRNA sequences. Curr Microbiol 56:391–396PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Jesús Morales-Jiménez
    • 1
    • 3
  • Gerardo Zúñiga
    • 2
  • Hugo C. Ramírez-Saad
    • 3
  • César Hernández-Rodríguez
    • 1
    Email author
  1. 1.Departamento de Microbiología, Escuela Nacional de Ciencias BiológicasInstituto Politécnico NacionalDistrito FederalMexico
  2. 2.Departamento de Zoología, Escuela Nacional de Ciencias BiológicasInstituto Politécnico NacionalDistrito FederalMexico
  3. 3.Laboratorio de Ecología Molecular, Departamento de Sistemas BiológicosUniversidad Autónoma Metropolitana—XochimilcoMéxicoMexico

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