Journal of Chemical Ecology

, Volume 41, Issue 1, pp 75–84 | Cite as

Aspen Defense Chemicals Influence Midgut Bacterial Community Composition of Gypsy Moth

  • Charles J. MasonEmail author
  • Kennedy F. Rubert-Nason
  • Richard L. Lindroth
  • Kenneth F. Raffa


Microbial symbionts are becoming increasingly recognized as mediators of many aspects of plant – herbivore interactions. However, the influence of plant chemical defenses on gut associates of insect herbivores is less well understood. We used gypsy moth (Lymantria dispar L.), and differing trembling aspen (Populus tremuloides Michx.) genotypes that vary in chemical defenses, to assess the influence of foliar chemistry on bacterial communities of larval midguts. We evaluated the bacterial community composition of foliage, and of midguts of larvae feeding on those leaves, using next-generation high-throughput sequencing. Plant defense chemicals did not influence the composition of foliar communities. In contrast, both phenolic glycosides and condensed tannins affected the bacterial consortia of gypsy moth midguts. The two most abundant operational taxonomic units were classified as Ralstonia and Acinetobacter. The relative abundance of Ralstonia was higher in midguts than in foliage when phenolic glycoside concentrations were low, but lower in midguts when phenolic glycosides were high. In contrast, the relative abundance of Ralstonia was lower in midguts than in foliage when condensed tannin concentrations were low, but higher in midguts when condensed tannins were high. Acinetobacter showed a different relationship with host chemistry, being relatively more abundant in midguts than with foliage when condensed tannin concentrations were low, but lower in midguts when condensed tannins were high. Acinetobacter tended to have a greater relative abundance in midguts of insects feeding on genotypes with high phenolic glycoside concentrations. These results show that plant defense chemicals influence herbivore midgut communities, which may in turn influence host utilization.


Bacteria Community Condensed tannins Midgut Phenolic glycosides Plant-insect interactions 



We thank Andrew Helm for assistance with analyzing condensed tannins. Critical reviews by Dr. Claudio Gratton and two anonymous referees, and editorial comments, improved this manuscript. This work was supported by USDA Hatch WIS#01598 awarded to K. Raffa, NSF grant DEB 0841609 to R. Lindroth, and the University of Wisconsin-Madison College of Agricultural and Life Sciences.

Supplementary material

10886_2014_530_Fig6_ESM.jpg (447 kb)
Supplemental Fig. 1

Sample accumulation curves of each sequenced sample in this study. Samples were subsampled prior to analysis. Leveling of curves indicate adequate depth of sequence. (JPEG 447 kb)

10886_2014_530_MOESM1_ESM.eps (193 kb)
High Resolution Image (EPS 193 kb)
10886_2014_530_MOESM2_ESM.docx (25 kb)
Supplemental Table 1 (DOCX 25 kb)


  1. Abreu IN, Ahnlund M, Moritz T, Albrectsen BR (2011) UHPLC-ESI/TOFMS determination of salicylate-like phenolic gycosides in Populus tremula leaves. J Chem Ecol 37:857–870PubMedCentralPubMedCrossRefGoogle Scholar
  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. Adams AS, Aylward FO, Adams SM, Erbilgin N, Aukema BH, Currie CR, Suen G, Raffa KF (2013) Mountain pine beetles colonizing historical and naive host trees are associated with a bacterial community highly enriched in genes contributing to terpene metabolism. Appl Environ Microbiol 79:3468–3475PubMedCentralPubMedCrossRefGoogle Scholar
  4. Anderson RC, Rasmussen MA, Allison MJ (1993) Metabolism of the plant toxins nitropropionic acid and nitropropanol by ruminal microorganisms. Appl Environ Microbiol 59:3056–3061PubMedCentralPubMedGoogle Scholar
  5. Appel HM, Maines LW (1995) The influence of host plant on gut conditions of gypsy moth (Lymantria dispar) caterpillars. J Insect Physiol 41:241–246CrossRefGoogle Scholar
  6. Ayayee P, Rosa C, Ferry JG, Felton GW, Saunders M, Hoover K (2014) Gut microbes contribute to nitrogen provisioning in a wood-feeding cerambycid. Environ Entomol 43(4):903–912Google Scholar
  7. Bailey JK, Deckert R, Schweitzer JA, Rehill BJ, Lindroth RL, Gehring C, Whitham TG (2005) Host plant genetics affect hidden ecological players: links among Populus, condensed tannins, and fungal endophyte infection. Can J Bot 83:356–361CrossRefGoogle Scholar
  8. Barbehenn RV, Constabel C (2011) Tannins in plant-herbivore interactions. Phytochemistry 72:1551–65PubMedCrossRefGoogle Scholar
  9. Barbehenn RV, Jaros A, Lee G, Mozola C, Weir Q, Salminen J-P (2009) Tree resistance to Lymantria dispar caterpillars: importance and limitations of foliar tannin composition. Oecologia 159:777–88PubMedCrossRefGoogle Scholar
  10. Boeckler GA, Gershenzon J, Unsicker SB (2011) Phenolic glycosides of the Salicaceae and their role as anti-herbivore defenses. Phytochemistry 72:1497–509, Elsevier LtdPubMedCrossRefGoogle Scholar
  11. Boone CK, Keefover-Ring K, Mapes AC, Adams AS, Bohlmann J, Raffa KF (2013) Bacteria associated with a tree-killing insect reduce concentrations of plant defense compounds. J Chem Ecol 39:1003–1006PubMedCrossRefGoogle Scholar
  12. Broderick NA, Raffa KF, Goodman RM, Handelsman J (2004) Census of the bacterial community of the gypsy moth larval midgut by using culturing and culture-independent methods. Appl Environ Microbiol 70:293–300PubMedCentralPubMedCrossRefGoogle Scholar
  13. Chung SH, Rosa C, Scully ED, Peiffer M, Tooker JF, Hoover K, Luthe DS, Felton GW (2013) Herbivore exploits orally secreted bacteria to suppress plant defenses. Proc Natl Acad Sci U S A 110:15728–15733PubMedCentralPubMedCrossRefGoogle Scholar
  14. Diguistini S, Wang Y, Liao NY, Taylor G, Tanguay P, Feau N, Henrissat B, Chan SK, Hesse-Orce U, Alamouti S, Tsui C, Docking R, Levasseur A, Haridas S, Roberston G, BIROL I, Holt R, Marra M, Hamelin R, Hirst M, Jones S, Bohlmann J, Breuil C (2011) Genome and transcriptome analyses of the mountain pine beetle-fungal symbiont Grosmannia clavigera, a lodgepole pine pathogen. Proc Natl Acad Sci U S A 108:2504–2509PubMedCentralPubMedCrossRefGoogle Scholar
  15. Donaldson JR, Stevens MT, Barnhill HR, Lindroth RL (2006) Age-related shifts in leaf chemistry of clonal aspen (Populus tremuloides). J Chem Ecol 32:1415–1429Google Scholar
  16. Dowd PF, Shen SK (1990) The contribution of symbiotic yeast to toxin resistance of the cigarette beetle (Lasioderma serricorne). Entomol Exp Appl 56:241–248CrossRefGoogle Scholar
  17. Driebe EM, Whitham TG (2000) Cottonwood hybridization affects tannin and nitrogen content of leaf litter and alters decomposition. Oecologia 123:99–107CrossRefGoogle Scholar
  18. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194–2200PubMedCentralPubMedCrossRefGoogle Scholar
  19. Geib SM, Filley TR, Hatcher PG, Hoover K, Carlson JE, Jimenez-Gasco MDM, Nakagawa-Izumi A, Sleighter RL, Tien M (2008) Lignin degradation in wood-feeding insects. Proc Natl Acad Sci U S A 105:12932–12937PubMedCentralPubMedCrossRefGoogle Scholar
  20. Gündüz EA, Douglas AE (2009) Symbiotic bacteria enable insect to use a nutritionally inadequate diet. Proc Biol Sci 276:987–991CrossRefGoogle Scholar
  21. Hagerman AE, Butler LG (1980) Condensed tannin purification and characterization of tannin-associated proteins. J Agric Food Chem 28:947–952PubMedCrossRefGoogle Scholar
  22. Hammerbacher A, Schmidt A, Wadke N, Wright LP, Schneider B, Bohlmann J, Brand WA, Fenning TM, Gershenzon J, Paetz C (2013) A common fungal associate of the spruce bark beetle metabolizes the stilbene defenses of Norway spruce. Plant Physiol 162:1324–1336PubMedCentralPubMedCrossRefGoogle Scholar
  23. Hanshew AS, Mason CJ, Raffa KF, Currie CR (2013) Minimization of chloroplast contamination in 16S rRNA gene pyrosequencing of insect herbivore bacterial communities. J Microbiol Methods 95:149–155PubMedCentralPubMedCrossRefGoogle Scholar
  24. Hemming JDC, Lindroth RL (1995) Intraspecific variation in aspen phytochemistry: effects on performance of gypsy moths and forest tent caterpillars. Oecologia 103:79–88CrossRefGoogle Scholar
  25. Holeski LM, Vogelzang A, Stanosz G, Lindroth RL (2009) Incidence of Venturia shoot blight in aspen (Populus tremuloides Michx.) varies with tree chemistry and genotype. Biochem Syst Ecol 37:139–145CrossRefGoogle Scholar
  26. Hosokawa T, Hironaka M, Mukai H, Inadomi K, Suzuki N, Fukatsu T (2012) Mothers never miss the moment: a fine-tuned mechanism for vertical symbiont transmission in a subsocial insect. Anim Behav 83:293–300CrossRefGoogle Scholar
  27. Humphrey PT, Nguyen TT, Villalobos MM, Whiteman NK (2014) Diversity and abundance of phyllosphere bacteria are linked to insect herbivory. Mol Ecol 23:1497–1515PubMedCrossRefGoogle Scholar
  28. Hwang S-Y, Lindroth RL (1997) Clonal variation in foliar chemistry of aspen: effects on gypsy moths and forest tent caterpillars. Oecologia 111:99–108CrossRefGoogle Scholar
  29. Johnson KS, Barbehenn RV (2000) Oxygen levels in the gut lumens of herbivorous insects. J Insect Physiol 46:897–903PubMedCrossRefGoogle Scholar
  30. Kaltenpoth M, Winter SA, Kleinhammer A (2009) Localization and transmission route of Coriobacterium glomerans, the endosymbiont of pyrrhocorid bugs. FEMS Microbiol Ecol 69:373–383PubMedCrossRefGoogle Scholar
  31. Kikuchi Y, Hosokawa T, Fukatsu T (2007) Insect-microbe mutualism without vertical transmission: a stinkbug acquires a beneficial gut symbiont from the environment every generation. Appl Environ Microbiol 73:4308–4316PubMedCentralPubMedCrossRefGoogle Scholar
  32. Kikuchi Y, Hayatsu M, Hosokawa T, Nagayama A, Tago K, Fukatsu T (2012) Symbiont-mediated insecticide resistance. Proc Natl Acad Sci U S A 109:8619–8622CrossRefGoogle Scholar
  33. Klepzig KD, Smalley EB, Raffa KF (1996) Combined chemical defenses against an insect-fungal complex. J Chem Ecol 22:1367–1388PubMedCrossRefGoogle Scholar
  34. Kohl KD, Dearing MD (2012) Experience matters: prior exposure to plant toxins enhances diversity of gut microbes in herbivores. Ecol Lett 15:1008–1015PubMedCrossRefGoogle Scholar
  35. Lees GL, Suttill NH, Gruber MY (1993) Condensed tannins in sainfoin. 1. A histological and cytological survey of plant tissues. Can J Bot 71:1147–1152CrossRefGoogle Scholar
  36. Liebhold AM, Gottschalk KW, Muzika RM, Montgomery ME, Young R, O’Day K, and Kelley, B (1995) Suitability of North American tree species to the gypsy moth: a summary of field and laboratory tests. U.S. Department of Agriculture Forest Service NE Forest Experimental Station General Technical Bulletin NE-211. U.S. Department of Agriculture, Washington, D.CGoogle Scholar
  37. Lindow SE, Brandl MT (2003) Microbiology of the phyllosphere. Appl Environ Microbiol 69:1875–1883PubMedCentralPubMedCrossRefGoogle Scholar
  38. Lindroth R, Hwang S-Y (1996) Diversity, redundancy, and multiplicity in chemical defense systems of aspen. In: Romeo J, Saunders J, Barbosa P (eds) Phytochemical diversity and redundancy in ecological interactions SE - 2. Springer, US, pp 25–56CrossRefGoogle Scholar
  39. Lindroth RL, St. Clair SB (2013) Adaptations of quaking aspen (Populus tremuloides Michx.) for defense against herbivores. For Ecol Manag 299:14–21, Elsevier B.VCrossRefGoogle Scholar
  40. Lindroth RL, Scriber JM, Hsia MTS (1986) “Differential responses of tiger swallowtail subspecies to secondary metabolites from tulip tree and quaking aspen.” Oecologia 70(1):13–19Google Scholar
  41. Łukasik P, Van Asch M, Guo H, Ferrari J, Godfray HCJ (2013) Unrelated facultative endosymbionts protect aphids against a fungal pathogen. Ecol Lett 16:214–218PubMedCrossRefGoogle Scholar
  42. Mason CJ, Raffa KF (2014) Acquisition and structuring of larval midgut bacterial communities in gypsy moth (Lepidoptera: Erebidae) larvae. Environ Entomol 43:594–604CrossRefGoogle Scholar
  43. Mason CJ, Couture JJ, Raffa KF (2014a) Plant-associated bacteria degrade plant defense chemicals and reduce their adverse effects on an insect defoliator. Oecologia 175:901–910PubMedCrossRefGoogle Scholar
  44. Mason CJ, Pfammatter JA, Holeski LM, Raffa KF (2014b) Foliar bacterial communities of trembling aspen in a common garden. Can J Microbiol. doi: 10.1139/cjm-2014-0362 PubMedGoogle Scholar
  45. Miller AW, Kohl KD, Dearing MD (2014) The gastrointestinal tract of the white-throated woodrat (Neotoma albigula) harbors distinct consortia of oxalate-degrading bacteria. Appl Environ Microbiol 80:1595–1601PubMedCentralPubMedCrossRefGoogle Scholar
  46. Mithöfer A, Boland W (2012) Plant defense against herbivores: chemical aspects. Annu Rev Plant Biol 63:431–450PubMedCrossRefGoogle Scholar
  47. Morales-Jiménez J, Vera-Ponce De León A, García-Domínguez A, Martínez-Romero E, Zúñiga G, Hernández-Rodríguez C (2013) Nitrogen-fixing and uricolytic bacteria associated with the gut of Dendroctonus rhizophagus and Dendroctonus valens (Curculionidae: Scolytinae). Microb Ecol 66:200–10PubMedCrossRefGoogle Scholar
  48. Noda S, Kitade O, Inoue T, Kawai M, Kanuka M, Hiroshima K, Hongoh Y, Constantino R, Uys V, Zhong J, Kudo T, Ohkuma M (2007) Cospeciation in the triplex symbiosis of termite gut protists (Pseudotrichonympha spp.), their hosts, and their bacterial endosymbionts. Mol Ecol 16:1257–1266PubMedCrossRefGoogle Scholar
  49. North RD, Jackson CW, Howse PE (1997) Evolutionary aspects of ant-fungus interactions in leaf-cutting ants. Trends Ecol Evol 12:386–389PubMedCrossRefGoogle Scholar
  50. Oliver KM, Russell JA, Moran NA, Hunter MS (2003) Facultative bacterial symbionts in aphids confer resistance to parasitic wasps. Proc Natl Acad Sci U S A 100:1803–1807PubMedCentralPubMedCrossRefGoogle Scholar
  51. Osier TL, Lindroth RL (2001) Effects of genotype, nutrient availability, and defoliation on aspen phytochemistry and insect performance. J Chem Ecol 27:1289–1313PubMedCrossRefGoogle Scholar
  52. Osier TL, Hwang S, Lindroth R (2000) Effects of phytochemical variation in quaking aspen Populus tremuloides clones on gypsy moth Lymantria dispar performance in the field and laboratory. Ecol Entomol 25:197–207CrossRefGoogle Scholar
  53. Payyavula RS, Babst BA, Nelsen MP, Harding SA, Tsai C-J (2009) Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures. BMC Plant Biol 9:151PubMedCentralPubMedCrossRefGoogle Scholar
  54. Porter L, Hrstich L, Chan B (1986) The converstion of procyanidins and propelphinidins to cyanidin and delphinidin. Phytochemistry 2:223–230Google Scholar
  55. Priya NG, Ojha A, Kajla MK, Raj A, Rajagopal R (2012) Host plant induced variation in gut bacteria of Helicoverpa armigera. PLoS ONE 7:e30768PubMedCrossRefGoogle Scholar
  56. R Core Team. 2013. R: A language and environment for statistical computing. Vienna, Austria.Google Scholar
  57. Scarborough C, Ferrari J, Godfray HCJ (2005) Aphid protected from pathogen by endosymbiont. Science 310:1781PubMedCrossRefGoogle Scholar
  58. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541PubMedCentralPubMedCrossRefGoogle Scholar
  59. Schloss PD, Gevers D, Westcott SL (2011) Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PLoS ONE 6:e27310PubMedCentralPubMedCrossRefGoogle Scholar
  60. Sellmer JC, Mccown BH, Haissig BE (1989) Shoot culture dynamics of six Populus clones. Tree Physiol 5:219–227PubMedCrossRefGoogle Scholar
  61. Shao Y, Arias-Cordero E, Guo H, Bartram S, Boland W (2014) In Vivo Pyro-SIP assessing active gut microbiota of the cotton leafworm, Spodoptera littoralis. PLoS ONE 9:e85948PubMedCentralPubMedCrossRefGoogle Scholar
  62. Sonowal R, Nandimath K, Kulkarni SS, Koushika SP, Nanjundiah V, Mahadevan S (2013) Hydrolysis of aromatic β-glucosides by non-pathogenic bacteria confers a chemical weapon against predators. Proc Biol Sci 280:20130721PubMedCentralPubMedCrossRefGoogle Scholar
  63. Vorholt JA (2012) Microbial life in the phyllosphere. Nat Rev Microbiol 10:828–840PubMedCrossRefGoogle Scholar
  64. Wang Q, Garrity GM, Tiedje JM, Cole JR (2007) Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Appl Environ Microbiol 73:5261–5267PubMedCentralPubMedCrossRefGoogle Scholar
  65. Wink M (1993) The plant vacuole: a multifunctional compartment. J Exp Bot 44:231–246Google Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Charles J. Mason
    • 1
    • 2
    Email author
  • Kennedy F. Rubert-Nason
    • 1
  • Richard L. Lindroth
    • 1
  • Kenneth F. Raffa
    • 1
  1. 1.Department of EntomologyUniversity of Wisconsin-MadisonMadisonUSA
  2. 2.345 Russell LaboratoriesUniversity of Wisconsin-MadisonMadisonUSA

Personalised recommendations