, Volume 76, Issue 3, pp 277–291 | Cite as

Spiroplasma dominates the microbiome of khapra beetle: comparison with a congener, effects of life stage and temperature

  • D. M. Wilches
  • R. A. Laird
  • P. G. Fields
  • P. Coghlin
  • K. D. FloateEmail author


Khapra beetle, Trogoderma granarium (Coleoptera: Dermestidae), is among the world’s most invasive and destructive pests of stored agricultural products. Its pest status is enhanced by the ability of the larvae to undergo diapause, which increases their tolerance to adverse conditions including insecticides and extreme temperatures. The ability of insects to tolerate extreme conditions can be influenced by their associated bacterial community (the microbiome). Understanding this relationship may lead to improved methods of pest control, but the microbiome of T. granarium is unknown. Here we use next-generation sequencing to address three main questions: 1) How similar are the microbiomes of the closely-related species T. granarium and T. variabile? 2) How does the microbiome change across life stage and physiological state? 3) How is the microbiome of adult T. granarium affected by extreme temperatures? Our results show that the core microbiomes of T. granarium and T. variabile are similar in composition. However, adults of former species have a microbiome dominated by Spiroplasma bacteria (99% of amplified sequences), whereas Spiroplasma in the latter species is almost absent (< 2%). The microbiome of T. granarium differs across life stage (feeding vs non-feeding life stages); its presence in eggs confirms the vertical transmission of Spiroplasma. High temperatures significantly reduced the relative abundance of Spiroplasma, but an effect of low temperatures on the microbiome of T. granarium was not detected. Given its dominance in a key pest species, further study of the interaction between Spiroplasma and its T. granarium host is warranted.


temperature tolerance quarantine stored-product pests Spiroplasma khapra beetle 



We thank Steve Perlman and Matt Ballinger (University of Victoria, Victoria, British Columbia, Canada) for efforts to identify the strain of Spiroplasma in khapra beetle, and for comments on an earlier draft of this paper. We thank Muhammad Sagheer (Grain Research Laboratory, University of Agriculture, Faisalabad, Pakistan) for providing us with live khapra beetles. This is AAFC Lethbridge Research and Development Centre Contribution No. 38717026.


  1. Abdelghany AY, Suthisut D, Fields PG (2015) The effect of diapause and cold acclimation on the cold-hardiness of the warehouse beetle, Trogoderma variabile (Coleoptera: Dermestidae). Can Entomol 147:158–168CrossRefGoogle Scholar
  2. Anbutsu H, Fukatsu T (2011) Spiroplasma as a model insect endosymbiont. Environ Microbiol Rep 3:144–153. CrossRefPubMedGoogle Scholar
  3. Anbutsu H, Goto S, Fukatsu T (2008) High and low temperatures differently affect infection density and vertical transmission of male-killing Spiroplasma symbionts in Drosophila hosts. Appl Environ Microbiol 74:6053–6059CrossRefPubMedPubMedCentralGoogle Scholar
  4. Anderson MJ (2001) A new method for non-parametric multivariate analysis of variance. Austral Ecol 26:32–46. CrossRefGoogle Scholar
  5. Ashworth JR (1993) The biology of Lasioderma serricorne. J Stored Prod Res 29:291–303CrossRefGoogle Scholar
  6. Bansal R, Hulbert SH, Reese JC, Whitworth RJ, Stuart JJ, Chen M-S (2014) Pyrosequencing reveals the predominance of Pseudomonadaceae in gut microbiome of a gall midge. Pathogens 3:459–472. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bell C, Wilson S, Banks H (1984) Studies on the toxicity of phosphine to tolerant stages of Trogoderma granarium Everts (Coleoptera: Dermestidae). J Stored Prod Res 20:111–117. CrossRefGoogle Scholar
  8. Bright M, Bulgheresi S (2010) A complex journey: transmission of microbial symbionts. Nat Rev Microbiol 8:218–230. CrossRefPubMedPubMedCentralGoogle Scholar
  9. Brumin M, Kontsedalov S, Ghanim M (2011) Rickettsia influences thermotolerance in the whitefly Bemisia tabaci B biotype. Insect Sci 18:57–66. CrossRefGoogle Scholar
  10. Buchner P (1965) Endosymbiosis of animals with plant microorganims. Interscience Publishers, New York 909 ppGoogle Scholar
  11. Burges DH (1962) Diapause, pest status and control of the Khapra beetle, Trogoderma granarium Everts. Ann Appl Biol 50:614–617. CrossRefGoogle Scholar
  12. Caporaso JG et al (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Ceja-Navarro JA et al (2015) Gut microbiota mediate caffeine detoxification in the primary insect pest of coffee. Nat Commun 6:7618. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Chao A (1984) Nonparametric estimation of the number of classes in a population Scandinavian. J Stats 11:265–270Google Scholar
  15. Chen W-J, Hsieh F-C, Hsu F-C, Tasy Y-F, Liu J-R, Shih M-C (2014) Characterization of an insecticidal toxin and pathogenicity of Pseudomonas taiwanensis against insects. PLoS Pathog 10:e1004288. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Colwell RK (2009) Biodiversity: concepts, patterns and measurement. In: Levin SA (ed) The Princeton guide to ecology. Princeton University Press, Princeton, pp 257–263Google Scholar
  17. Dematheis F, Kurtz B, Vidal S, Smalla K (2012) Microbial communities associated with the larval gut and eggs of the western corn rootworm. PLoS One 7:e44685. CrossRefPubMedPubMedCentralGoogle Scholar
  18. DeSantis TZ et al (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72:5069–5072. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Di Bella JM, Bao Y, Gloor GB, Burton JP, Reid G (2013) High throughput sequencing methods and analysis for microbiome research. J Microbiol Methods 95:401–414. CrossRefPubMedGoogle Scholar
  20. Douglas AE (1989) Mycetocyte symbiosis in insects. Biol Rev 64:409–434. CrossRefPubMedGoogle Scholar
  21. Dowd PF (1989) In situ production of hydrolytic detoxifying enzymes by symbiotic yeasts in the cigarette beetle (Coleoptera: Anobiidae). J Econ Entomol 82:396–400. CrossRefGoogle Scholar
  22. Duron O, Bouchon D, Boutin S, Bellamy L, Zhou L, Engelstadter J, Hurst G (2008) The diversity of reproductive parasites among arthropods: Wolbachia do not walk alone. BMC Biol 6:27. CrossRefPubMedPubMedCentralGoogle Scholar
  23. Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:2460–2461CrossRefGoogle Scholar
  24. Eliopoulos P (2013) New approaches for tackling the khapra beetle. CAB Int Rev 8:1–13. CrossRefGoogle Scholar
  25. Engel P, Moran NA (2013) The gut microbiota of insects–diversity in structure and function. FEMS Microbiol Rev 37:699–735. CrossRefPubMedGoogle Scholar
  26. Enigl M, Schausberger P (2007) Incidence of the endosymbionts Wolbachia, Cardinium and Spiroplasma in phytoseiid mites and associated prey. Exp Appl Acarol 42:75–85. CrossRefPubMedGoogle Scholar
  27. Faith DP (1992) Conservation evaluation and phylogenetic diversity. Biol Conserv 61:1–10. CrossRefGoogle Scholar
  28. Feldhaar H, Gross R (2009) Insects as hosts for mutualistic bacteria. Int J Med Microbiol 299:1–8CrossRefPubMedGoogle Scholar
  29. Fischer M et al (2016) Efficacy assessment of nucleic acid decontamination reagents used in molecular diagnostic laboratories. PLoS One 11:e0159274. CrossRefPubMedPubMedCentralGoogle Scholar
  30. Fukatsu T et al (2007) Bacterial endosymbiont of the slender pigeon louse, Columbicola columbae, allied to endosymbionts of grain weevils and tsetse flies. Appl Environ Microbiol 73:6660–6668. CrossRefPubMedPubMedCentralGoogle Scholar
  31. Gasparich GE (2010) Spiroplasmas and phytoplasmas: Microbes associated with plant hosts. Biologicals 38:193–203. CrossRefPubMedGoogle Scholar
  32. Gasparich GE, Whitcomb RF, Dodge D, French FE, Glass J, Williamson DL (2004) The genus Spiroplasma and its non-helical descendants: Phylogenetic classification, correlation with phenotype and roots of the Mycoplasma mycoides clade. Int J Syst Evol Microbiol 54:893–918. CrossRefPubMedGoogle Scholar
  33. Goodacre SL, Martin OY, Thomas CFG, Hewitt GM (2006) Wolbachia and other endosymbiont infections in spiders. Mol Ecol 15:517–527. CrossRefPubMedGoogle Scholar
  34. Gosalbes MJ, Latorre A, Lamelas A, Moya A (2010) Genomics of intracellular symbionts in insects. Int J Med Microbiol 300:271–278. CrossRefPubMedGoogle Scholar
  35. Hail D, Dowd SE, Bextine B (2012) Identification and location of symbionts associated with potato psyllid (Bactericera cockerelli) lifestages. Environ Entomol 41:98–107. CrossRefPubMedGoogle Scholar
  36. Hammer TJ, McMillan WO, Fierer N (2014) Metamorphosis of a butterfly-associated bacterial community. PLoS One 9:e86995. CrossRefPubMedPubMedCentralGoogle Scholar
  37. Herren JK, Paredes JC, Schüpfer F, Lemaitre B (2013) Vertical transmission of a Drosophila endosymbiont via cooption of the yolk transport and internalization machinery. MBio 4:e00532–e00512CrossRefPubMedPubMedCentralGoogle Scholar
  38. Hugher A (1956) Experimentelle untersuchungen über die künstliche symbiontenelimination bei vorratsschädlingen: Rhizopertha dominica f. (Bostrychidae) und Oryzaephilus surinamensis l. (Cucujidae). Z Morphol Okol Tiere 44:626–701CrossRefGoogle Scholar
  39. Hughes JB, Hellmann JJ, Ricketts TH, Bohannan BJM (2001) Counting the uncountable: statistical approaches to estimating microbial diversity. Appl Environ Microbiol 67:4399–4406. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Jaenike J, Stahlhut JK, Boelio LM, Unckless RL (2010a) Association between Wolbachia and Spiroplasma within Drosophila neotestacea: an emerging symbiotic mutualism? Mol Ecol 19:414–425. CrossRefPubMedGoogle Scholar
  41. Jaenike J, Unckless R, Cockburn SN, Boelio LM, Perlman SJ (2010b) Adaptation via symbiosis: recent spread of a Drosophila defensive symbiont. Science 329:212–215. CrossRefPubMedGoogle Scholar
  42. Jiggins FM, Hurst GDD, Jiggins CD, vd Schulenburg JHG, Majerus MEN (2000) The butterfly Danaus chrysippus is infected by a male-killing Spiroplasma bacterium. Parasitology 120:439–446CrossRefPubMedGoogle Scholar
  43. Kageyama D, Narita S, Imamura T, Miyanoshita A (2010) Detection and identification of Wolbachia endosymbionts from laboratory stocks of stored-product insect pests and their parasitoids. J Stored Prod Res 46:13–19. CrossRefGoogle Scholar
  44. Kautz S, Rubin BER, Russell JA, Moreau CS (2013) Surveying the microbiome of ants: comparing 454 pyrosequencing with traditional methods to uncover bacterial diversity. Appl Environ Microbiol 79:525–534. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Koch A (1956) The experimental elimination of symbionts and its consequences. Exp Parasitol 5:481–518CrossRefPubMedGoogle Scholar
  46. Koštál V (2006) Eco-physiological phases of insect diapause. J Insect Physiol 52:113–127CrossRefPubMedGoogle Scholar
  47. Landesman WJ, Nelson DM, Fitzpatrick MC (2014) Soil properties and tree species drive ß-diversity of soil bacterial communities. Soil Biol Biochem 76:201–209. CrossRefGoogle Scholar
  48. Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic Acid Techniques in Bacterial Systematics. John Wiley and Sons, Chichester, pp 115–175Google Scholar
  49. Li YY, Floate KD, Fields PG, Pang BP (2014) Review of treatment methods to remove Wolbachia bacteria from arthropods. Symbiosis 62:1–15. CrossRefGoogle Scholar
  50. Li Y-Y, Fields P, Pang B-P, Coghlin P, Floate K (2015) Prevalence and diversity of Wolbachia bacteria infecting insect pests of stored products. J Stored Prod Res 62:93–100. CrossRefGoogle Scholar
  51. Lindgren DL, Vincent LE (1959) Biology and control of Trogoderma granarium Everts. J Econ Entomol 52:312–319. CrossRefGoogle Scholar
  52. Lindgren DL, Vincent LE, Krohne H (1955) The khapra beetle, Trogoderma granarium Everts Hilgardia 24:1–36CrossRefGoogle Scholar
  53. Loschiavo S (1960) Life-history and behaviour of Trogoderma parabile Beal (Coleoptera: Dermestidae). Can Entomol 92:611–618. CrossRefGoogle Scholar
  54. Lozupone C, Knight R (2005) UniFrac: a new phylogenetic method for comparing microbial communities. Appl Environ Microbiol 71:8228–8235. CrossRefPubMedPubMedCentralGoogle Scholar
  55. Majerus ME, Hinrich J, Schulenburg GV, Zakharov IA (2000) Multiple causes of male-killing in a single sample of the two-spot ladybird, Adalia bipunctata (Coleoptera: Coccinellidae) from Moscow. Heredity 84:605–609CrossRefPubMedGoogle Scholar
  56. Martin JD, Mundt JO (1972) Enterococci in insects. Appl Microbiol 24:575–580PubMedPubMedCentralGoogle Scholar
  57. Meriweather M, Matthews S, Rio R, Baucom RS (2013) A 454 survey reveals the community composition and core microbiome of the common bed bug (Cimex lectularius) across an urban landscape. PLoS One 8:e61465. CrossRefPubMedPubMedCentralGoogle Scholar
  58. Moll RM, Romoser WS, Modrakowski MC, Moncayo AC, Lerdthusnee K (2001) Meconial peritrophic membranes and the fate of midgut bacteria during mosquito (Diptera: Culicidae) metamorphosis. J Med Entomol 38:29–32. CrossRefPubMedGoogle Scholar
  59. Montenegro H, Solferini VN, Klaczko LB, Hurst GD (2005) Male-killing Spiroplasma naturally infecting Drosophila melanogaster. Insect Mol Biol 14:281–287CrossRefPubMedGoogle Scholar
  60. Musa AK, Dike MC (2009) Life cycle, morphometrics and damage assessment of the Khapra beetle, Trogoderma granarium Everts (Coleoptera: Dermestidae) on stored groundnut. J Agric Sci 54:135–142Google Scholar
  61. Neelakanta G, Sultana H, Fish D, Anderson J, Fikrig E (2010) Anaplasma phagocytophilum induces Ixodes scapularis ticks to express an antifreeze glycoprotein gene that enhances their survival in the cold. J Clin Invest 120:3179–3190. CrossRefPubMedPubMedCentralGoogle Scholar
  62. Nubel U, Garcia-Pichel F, Muyzer G (2000) The halotolerance and phylogeny of cyanobacteria with tightly coiled trichomes (Spirulina turpin) and the description of Halospirulina tapeticola gen. nov., sp. nov. Int J Syst Evol Microbiol 50:1265–1277. CrossRefPubMedGoogle Scholar
  63. OEPP/EPPO (2013a) Data sheets on quarantine pests No. 121: Trogoderma granarium Bulletin OEPP/EPPO Bulletin 11 doi:
  64. OEPP/EPPO (2013b) Diagnostics: Trogoderma granarium. Eur Mediterranean Plant Protection Organ Bull 43:431–448. CrossRefGoogle Scholar
  65. OEPP/EPPO (2015) EPPO A1 and A2 lists of pests recommended for regulation as quarantine pests EPPO Standards. Accessed 20 March 2018
  66. Opota O et al (2011) Monalysin, a novel ß-pore-forming toxin from the Drosophila pathogen Pseudomonas entomophila. In: contributes to host intestinal damage and lethality PLoS Pathog 7:e1002259Google Scholar
  67. Palavesam A et al (2012) Pyrosequencing-based analysis of the microbiome associated with the horn fly, Haematobia irritans. PLoS One 7:e44390. CrossRefPubMedPubMedCentralGoogle Scholar
  68. Pollock J, Glendinning L, Wisedchanwet T, Watson M (2018) The madness of microbiome: Attempting to find consensus “best practice” for 16S microbiome studies. Appl Environ Microbiol (accepted).
  69. Prince AM, Andrus L (1992) PCR: how to kill unwanted DNA. BioTechniques 12:358–360PubMedGoogle Scholar
  70. Rajendran S, Hajira Parveen KM (2005) Insect infestation in stored animal products. J Stored Prod Res 41:1–30. CrossRefGoogle Scholar
  71. R-Development-Core-Team (2011) R: A language and environment for statistical computing. R Foundation for Statistical Computing.
  72. Regassa LB, Gasparich GE (2006) Spiroplasmas: evolutionary relationships and biodiversity Front Biosci 11:2983–3002CrossRefPubMedGoogle Scholar
  73. Reid NM, Addison SL, Macdonald LJ, Lloyd-Jones G (2011) Biodiversity of active and inactive bacteria in the gut flora of wood-feeding Huhu beetle larvae (Prionoplus reticularis). Appl Environ Microbiol 77:7000–7006. CrossRefPubMedPubMedCentralGoogle Scholar
  74. Robinson C, Schloss P, Ramos Y, Raffa K, Handelsman J (2010) Robustness of the bacterial community in the cabbage white butterfly larval midgut. Microb Ecol 59:199–211. CrossRefPubMedGoogle Scholar
  75. Rogers EE, Backus EA (2014) Anterior foregut microbiota of the glassy-winged sharpshooter explored using deep 16S rRNA gene sequencing from individual insects. PLoS One 9:e106215. CrossRefPubMedPubMedCentralGoogle Scholar
  76. Russell JA, Moran NA (2006) Costs and benefits of symbiont infection in aphids: variation among symbionts and across temperatures. Proc R Soc Lond B Bio 273:603–610CrossRefGoogle Scholar
  77. Sachs JL, Essenberg CJ, Turcotte MM (2011) New paradigms for the evolution of beneficial infections. Trends Ecol Evol 26:202–209CrossRefPubMedGoogle Scholar
  78. Salter SJ, Cox MJ, Turek EM, Calus ST, Cookson WO, Moffatt MF, Turner P, Parkhill J, Loman NJ, Walker AW (2014) Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol 12:87CrossRefPubMedPubMedCentralGoogle Scholar
  79. Shade A, Handelsman J (2012) Beyond the Venn diagram: the hunt for a core microbiome. Environ Microbiol 14:4–12. CrossRefPubMedGoogle Scholar
  80. Shannon CE, Weaver W (1949) The mathematical theory of communication. University of Illinois Press, UrbanaGoogle Scholar
  81. Tagliavia M, Messina E, Manachini B, Cappello S, Quatrini P (2014) The gut microbiota of larvae of Rhynchophorus ferrugineus Oliver (Coleoptera: Curculionidae). BMC Microbiol 14:136–136. CrossRefPubMedPubMedCentralGoogle Scholar
  82. Tauber MJ, Tauber CA, Masaki S (1986) The diapause syndrome. In: Seasonal adaptations of insects. Oxford University Press, New York, pp 67–106Google Scholar
  83. Trivedi J, Srivastava A, Narain K, Chatterjee R (1991) The digestion of wool fibres in the alimentary system of Anthrenus flavipes larvae. Int Biodeterior 27:327–336CrossRefGoogle Scholar
  84. Vodovar N et al (2006) Complete genome sequence of the entomopathogenic and metabolically versatile soil bacterium Pseudomonas entomophila. Nat Biotechnol 24:673–679CrossRefPubMedGoogle Scholar
  85. Wang Y, Zhang Y (2015) Investigation of gut-associated bacteria in Tenebrio molitor (Coleoptera: Tenebrionidae) larvae using culture-dependent and DGGE methods. Ann Entomol Soc Am 108:941–949. CrossRefGoogle Scholar
  86. Welch EW, Macias J, Bextine B (2015) Geographic patterns in the bacterial microbiome of the glassy-winged sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae). Symbiosis 66:1–12. CrossRefGoogle Scholar
  87. Wernegreen JJ (2012) Mutualism meltdown in insects: bacteria constrain thermal adaptation. Curr Opin Microbiol 15:255–262. CrossRefPubMedPubMedCentralGoogle Scholar
  88. Wilches DM, Laird RA, Floate KD, Fields PG (2016) A review of diapause and tolerance to extreme temperatures in dermestids (Coleoptera). J Stored Prod Res 68:50–62. CrossRefGoogle Scholar
  89. Wilches DM, Laird RA, Floate KD, Fields PG (2017) Effects of acclimation and diapause on the cold tolerance of Trogoderma granarium. Entomol Exp Appl 165:169–178. CrossRefGoogle Scholar
  90. Xie J, Vilchez I, Mateos M (2010) Spiroplasma bacteria enhance survival of Drosophila hydei attacked by the parasitic wasp Leptopilina heterotoma. PLoS One 5:e12149. CrossRefPubMedPubMedCentralGoogle Scholar
  91. Zchori-Fein E, Perlman SJ (2004) Distribution of the bacterial symbiont Cardinium in arthropods. Mol Ecol 13:2009–2016. CrossRefPubMedGoogle Scholar
  92. Zhantiev RD (2009) Ecology and classification of dermestid beetles (Coleoptera: Dermestidae) of the Palaearctic fauna. Entomol Rev 89:157–174. CrossRefGoogle Scholar
  93. Zindel R, Gottlieb Y, Aebi A (2011) Arthropod symbioses: A neglected parameter in pest- and disease-control programmes. J Appl Ecol 48:864–872CrossRefGoogle Scholar

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© Crown 2018

Authors and Affiliations

  1. 1.Lethbridge Research and Development CentreAgriculture and Agri-Food CanadaLethbridgeCanada
  2. 2.Department of Biological SciencesUniversity of LethbridgeLethbridgeCanada
  3. 3.Morden Research and Development CentreAgriculture and Agri-Food CanadaWinnipegCanada

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