Microbial Ecology

, Volume 78, Issue 1, pp 206–222 | Cite as

Distribution and Variation of Bacterial Endosymbiont and “Candidatus Liberibacter asiaticus” Titer in the Huanglongbing Insect Vector, Diaphorina citri Kuwayama

  • Saeed Hosseinzadeh
  • Masoud Shams-BakhshEmail author
  • Marina Mann
  • Somayeh Fattah-Hosseini
  • Abdoolnabi Bagheri
  • Mohammad Mehrabadi
  • Michelle HeckEmail author
Invertebrate Microbiology


The Asian citrus psyllid (ACP), Diaphorina citri Kuwayama, is an economic insect pest in most citrus-growing regions and the vector of ‘Candidatus Liberibacter asiaticus’ (CLas), one of at least three known bacteria associated with Huanglongbing (HLB or citrus greening disease). D. citri harbors bacterial endosymbionts, including Wolbachia pipientis (strain Wolbachia wDi), ‘Candidatus Carsonella ruddii,’ and ‘Candidatus Profftella armatura.’ Many important functions of these bacteria can be inferred from their genome sequences, but their interactions with each other, CLas, and their D. citri host are poorly understood. In the present study, the titers of the endosymbionts in different tissues, in each sex, and in insects reared on healthy citrus (referred to as unexposed) and CLas-infected citrus (referred to as CLas-exposed) D. citri were investigated using real-time, quantitative PCR (qPCR) using two different quantitative approaches. Wolbachia and CLas were detected in all insect tissues. The titer of Wolbachia was higher in heads of CLas-exposed males as compared to unexposed males. In males and females, Wolbachia titer was highest in the Malpighian tubules. The highest titer of CLas was observed in the gut. Profftella and Carsonella titers were significantly reduced in the bacteriome of CLas-exposed males compared with that of unexposed males, but this effect was not observed in females. In ovaries of CLas-exposed females, the Profftella and Carsonella titers were increased as compared to non-exposed females. CLas appeared to influence the overall levels of the symbionts but did not drastically perturb the overall microbial community structure. In all the assessed tissues, CLas titer in males was significantly higher than that of females using absolute quantification. These data provide a better understanding of multi-trophic interactions regulating symbiont dynamics in the HLB pathosystem.


Huanglongbing Wolbachia pipientis Candidatus Carsonella ruddii” Candidatus Profftella armatura” Diaphorina citri Candidatus Liberibacter asiaticus” 



Funding for the study was provided by the California Citrus Research Board grant numbers 5300-155 and 5300-163 (to MH) and the USDA NIFA SCRI grant number 8062-22410-006-45-I (to MH). We thank three anonymous reviewers for their thoughtful and constructive feedback.

Supplementary material

248_2018_1290_Fig8_ESM.png (922 kb)
Figure S1

Different tissues of D. citri that were dissected and used in this study. Dissections occurred under natural light in 1× PBST. a Side view of an adult female D. citri. b Side view of an adult male D. citri. c Whole gut with Malpighian tubules. d Separated head attached to salivary glands. e Gut after removing the Malpighian tubules. f Separated Malpighian tubules. g Bacteriome. h Ovary. i Male reproductive system (scale bar = 200 μm). (PNG 922 kb)

248_2018_1290_MOESM1_ESM.tif (2 mb)
High-Resolution Image (TIF 2080 kb)


  1. 1.
    Gottwald TR (2010) Current epidemiological understanding of citrus huanglongbing. Annu Rev Phytopathol 48:119–139CrossRefGoogle Scholar
  2. 2.
    Bové J (2006) Huanglongbing: a destructive, newly-emerging, century-old disease of citrus [Asia; South Africa; Brazil; Florida]. J Plant PatholGoogle Scholar
  3. 3.
    da Graça JV, Douhan GW, Halbert SE, Keremane ML, Lee RF, Vidalakis G, Zhao H (2016) Huanglongbing: an overview of a complex pathosystem ravaging the world's citrus. J Integr Plant Biol 58(4):373–387CrossRefGoogle Scholar
  4. 4.
    Van der Merwe A (1937) Chromium and manganese toxicity. Is it important in Transvaal citrus greening? Farming S Afr 12:439–440Google Scholar
  5. 5.
    Texeira DdC, Ayres J, Kitajima EW, Danet L, Jagoueix-Eveillard S, Saillard C, Bové JM (2005) First report of a huanglongbing-like disease of citrus in São Paulo State, Brazil and association of a new Liberibacter species,“Candidatus Liberibacter americanus”, with the disease. Plant Dis 89(1):107–107CrossRefGoogle Scholar
  6. 6.
    Halbert S (2005) The discovery of huanglongbing in Florida. in Proceedings of the international citrus canker and huanglongbing research workshop, Orlando, H-3Google Scholar
  7. 7.
    Lopes S et al (2013) HLB research in Brazil—from etiology to disease management. in Proceedings of the 19th conference IOCVGoogle Scholar
  8. 8.
    Luis M et al (2009) Occurrence of citrus huanglongbing in Cuba and association of the disease with Candidatus Liberibacter asiaticus. J Plant Pathol:709–712Google Scholar
  9. 9.
    Manjunath K et al (2010) First report of the citrus huanglongbing associated bacterium ‘Candidatus Liberibacter asiaticus’ from sweet orange, Mexican lime, and Asian citrus psyllid in Belize. Plant Dis 94(6):781–781CrossRefGoogle Scholar
  10. 10.
    Trujillo-Arriga J et al (2010) Antecedantes y situación actual de Huanglongbing de los cítricos en México. in Memoria 1er Simposio Nacional Sobre Investigación para el Manejo del Psílido Asiático de los Cítricos y el HLB en MéxicoGoogle Scholar
  11. 11.
    Faghihi M et al (2009) First report of citrus huanglongbing disease on orange in Iran. Plant Pathol 58(4):793–793CrossRefGoogle Scholar
  12. 12.
    Inoue H et al (2009) Enhanced proliferation and efficient transmission of Candidatus Liberibacter asiaticus by adult Diaphorina citri after acquisition feeding in the nymphal stage. Ann Appl Biol 155(1):29–36CrossRefGoogle Scholar
  13. 13.
    Hall DG, Richardson ML, Ammar ED, Halbert SE (2013) Asian citrus psyllid, Diaphorina citri, vector of citrus huanglongbing disease. Entomol Exp Appl 146(2):207–223CrossRefGoogle Scholar
  14. 14.
    Bonani J et al (2010) Characterization of electrical penetration graphs of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings. Entomol Exp Appl 134(1):35–49CrossRefGoogle Scholar
  15. 15.
    Pelz-Stelinski K, Killiny N (2016) Better together: association with ‘Candidatus Liberibacter asiaticus’ increases the reproductive fitness of its insect vector, Diaphorina citri (Hemiptera: Liviidae). Ann Entomol Soc Am 109(3):371–376CrossRefGoogle Scholar
  16. 16.
    Chu C-C, Gill TA, Hoffmann M, Pelz-Stelinski KS (2016) Inter-population variability of endosymbiont densities in the Asian citrus psyllid (Diaphorina citri Kuwayama). Microb Ecol 71(4):999–1007CrossRefGoogle Scholar
  17. 17.
    Ghanim M, Fattah-Hosseini S, Levy A, Cilia M (2016) Morphological abnormalities and cell death in the Asian citrus psyllid (Diaphorina citri) midgut associated with Candidatus Liberibacter asiaticus. Sci Rep 6:33418CrossRefGoogle Scholar
  18. 18.
    Nakabachi A, Ueoka R, Oshima K, Teta R, Mangoni A, Gurgui M, Oldham NJ, van Echten-Deckert G, Okamura K, Yamamoto K, Inoue H, Ohkuma M, Hongoh Y, Miyagishima SY, Hattori M, Piel J, Fukatsu T (2013) Defensive bacteriome symbiont with a drastically reduced genome. Curr Biol 23(15):1478–1484CrossRefGoogle Scholar
  19. 19.
    Saha S, Hunter WB, Reese J, Morgan JK, Marutani-Hert M, Huang H, Lindeberg M (2012) Survey of endosymbionts in the Diaphorina citri metagenome and assembly of a Wolbachia wDi draft genome. PLoS One 7(11):e50067CrossRefGoogle Scholar
  20. 20.
    Nakabachi A, Yamashita A, Toh H, Ishikawa H, Dunbar HE, Moran NA, Hattori M (2006) The 160-kilobase genome of the bacterial endosymbiont Carsonella. Science 314(5797):267–267CrossRefGoogle Scholar
  21. 21.
    Tamames J, Gil R, Latorre A, Peretó J, Silva FJ, Moya A (2007) The frontier between cell and organelle: genome analysis of Candidatus Carsonella ruddii. BMC Evol Biol 7(1):181CrossRefGoogle Scholar
  22. 22.
    Ramsey JS, Johnson RS, Hoki JS, Kruse A, Mahoney J, Hilf ME, Hunter WB, Hall DG, Schroeder FC, MacCoss MJ, Cilia M (2015) Metabolic interplay between the Asian citrus psyllid and its Profftella symbiont: an Achilles’ heel of the citrus greening insect vector. PLoS One 10(11):e0140826CrossRefGoogle Scholar
  23. 23.
    Nakabachi A, Nikoh N, Oshima K, Inoue H, Ohkuma M, Hongoh Y, Miyagishima SY, Hattori M, Fukatsu T (2013) Horizontal gene acquisition of Liberibacter plant pathogens from a bacteriome-confined endosymbiont of their psyllid vector. PLoS One 8(12):e82612CrossRefGoogle Scholar
  24. 24.
    Fagen JR, Giongo A, Brown CT, Davis-Richardson AG, Gano KA, Triplett EW (2012) Characterization of the relative abundance of the citrus pathogen Ca. Liberibacter asiaticus in the microbiome of its insect vector, Diaphorina citri, using high throughput 16S rRNA sequencing. The Open Microbiology Journal 6:29–33CrossRefGoogle Scholar
  25. 25.
    Jain M, Fleites LA, Gabriel DW (2017) A small wolbachia protein directly represses phage lytic cycle genes in “Candidatus Liberibacter asiaticus” within psyllids. mSphere 2(3):e00171–e00117CrossRefGoogle Scholar
  26. 26.
    Mann M et al (2018) Diaphorina citri nymphs are resistant to morphological changes induced by “Candidatus Liberibacter asiaticus” in midgut epithelial cells. Infect Immun p. IAI. 00889-17Google Scholar
  27. 27.
    Hoffmann M, Coy MR, Gibbard HNK, Pelz-Stelinski KS (2014) Wolbachia infection density in populations of the Asian citrus psyllid (Hemiptera: Liviidae). Environ Entomol 43(5):1215–1222CrossRefGoogle Scholar
  28. 28.
    Kondo N, Shimada M, Fukatsu T (2005) Infection density of Wolbachia endosymbiont affected by co-infection and host genotype. Biol Lett 1(4):488–491CrossRefGoogle Scholar
  29. 29.
    Engel P, Moran NA (2013) The gut microbiota of insects—diversity in structure and function. FEMS Microbiol Rev 37(5):699–735CrossRefGoogle Scholar
  30. 30.
    Dossi FCA, da Silva EP, Cônsoli FL (2014) Population dynamics and growth rates of endosymbionts during Diaphorina citri (Hemiptera, Liviidae) ontogeny. Microb Ecol 68(4):881–889CrossRefGoogle Scholar
  31. 31.
    Godornes C, Leader BT, Molini BJ, Centurion-Lara A, Lukehart SA (2007) Quantitation of rabbit cytokine mRNA by real-time RT-PCR. Cytokine 38(1):1–7CrossRefGoogle Scholar
  32. 32.
    Li W, Hartung JS, Levy L (2006) Quantitative real-time PCR for detection and identification of Candidatus Liberibacter species associated with citrus huanglongbing. J Microbiol Methods 66(1):104–115CrossRefGoogle Scholar
  33. 33.
    Tiwari S, Lewis-Rosenblum H, Pelz-Stelinski K, Stelinski LL (2010) Incidence of Candidatus Liberibacter asiaticus infection in abandoned citrus occurring in proximity to commercially managed groves. J Econ Entomol 103(6):1972–1978CrossRefGoogle Scholar
  34. 34.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25(4):402–408CrossRefGoogle Scholar
  35. 35.
    Kliot A et al (2014) Fluorescence in situ hybridizations (FISH) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues. J Vis Exp: JoVE (84)Google Scholar
  36. 36.
    Heddi A, Grenier AM, Khatchadourian C, Charles H, Nardon P (1999) Four intracellular genomes direct weevil biology: nuclear, mitochondrial, principal endosymbiont, and Wolbachia. Proc Natl Acad Sci 96(12):6814–6819CrossRefGoogle Scholar
  37. 37.
    Pelz-Stelinski K et al (2010) Transmission parameters for Candidatus Liberibacter asiaticus by Asian citrus psyllid (Hemiptera: Psyllidae). J Econ Entomol 103(5):1531–1541CrossRefGoogle Scholar
  38. 38.
    Mann RS, Pelz-Stelinski K, Hermann SL, Tiwari S, Stelinski LL (2011) Sexual transmission of a plant pathogenic bacterium, Candidatus Liberibacter asiaticus, between conspecific insect vectors during mating. PLoS One 6(12):e29197CrossRefGoogle Scholar
  39. 39.
    Koga R, Tsuchida T, Fukatsu T (2003) Changing partners in an obligate symbiosis: a facultative endosymbiont can compensate for loss of the essential endosymbiont Buchnera in an aphid. Proc R Soc Lond B Biol Sci 270(1533):2543–2550CrossRefGoogle Scholar
  40. 40.
    Veneti Z, Clark ME, Karr TL, Savakis C, Bourtzis K (2004) Heads or tails: host-parasite interactions in the Drosophila-Wolbachia system. Appl Environ Microbiol 70(9):5366–5372CrossRefGoogle Scholar
  41. 41.
    Dyer KA, Minhas MD, Jaenike J (2005) Expression and modulation of embryonic male-killing in Drosophila innubila: opportunities for multilevel selection. Evolution 59(4):838–848CrossRefGoogle Scholar
  42. 42.
    Ammar E-D, Shatters Jr RG, Lynch C, Hall DG (2011) Detection and relative titer of Candidatus Liberibacter asiaticus in the salivary glands and alimentary canal of Diaphorina citri (Hemiptera: Psyllidae) vector of citrus huanglongbing disease. Ann Entomol Soc Am 104(3):526–533CrossRefGoogle Scholar
  43. 43.
    Ammar ED, Shatters Jr RG, Hall DG (2011) Localization of Candidatus Liberibacter asiaticus, associated with citrus huanglongbing disease, in its psyllid vector using fluorescence in situ hybridization. J Phytopathol 159(11–12):726–734CrossRefGoogle Scholar
  44. 44.
    Ammar E-D, Hall DG, Hosseinzadeh S, Heck M (2018) The quest for a non-vector psyllid: natural variation in acquisition and transmission of the huanglongbing pathogen ‘Candidatus Liberibacter asiaticus’ by Asian citrus psyllid isofemale lines. PLoS One 13(4):e0195804CrossRefGoogle Scholar
  45. 45.
    Huang J et al (2015) Studies on the relationship between feeding sites and bacterium acquisition efficiency of Diaphorina citri on Huanglongbing-infected citrus. J South China Agr Univ 36(1):71–74Google Scholar
  46. 46.
    Sétamou M, Simpson CR, Alabi OJ, Nelson SD, Telagamsetty S, Jifon JL (2016) Quality matters: influences of citrus flush physicochemical characteristics on population dynamics of the Asian citrus psyllid (Hemiptera: Liviidae). PLoS One 11(12):e0168997CrossRefGoogle Scholar
  47. 47.
    Ammar E-D, Ramos JE, Hall DG, Dawson WO, Shatters RG (2016) Acquisition, replication and inoculation of Candidatus Liberibacter asiaticus following various acquisition periods on huanglongbing-infected citrus by nymphs and adults of the Asian citrus psyllid. PLoS One 11(7):e0159594CrossRefGoogle Scholar
  48. 48.
    Nault L (1997) Arthropod transmission of plant viruses: a new synthesis. Ann Entomol Soc Am 90(5):521–541CrossRefGoogle Scholar
  49. 49.
    Vyas M, Fisher TW, He R, Nelson W, Yin G, Cicero JM, Willer M, Kim R, Kramer R, May GA, Crow JA, Soderlund CA, Gang DR, Brown JK (2015) Asian citrus psyllid expression profiles suggest Candidatus Liberibacter asiaticus-mediated alteration of adult nutrition and metabolism, and of nymphal development and immunity. PLoS One 10(6):e0130328CrossRefGoogle Scholar
  50. 50.
    Yan Q, Sreedharan A, Wei S, Wang J, Pelz-Stelinski K, Folimonova S, Wang N (2013) Global gene expression changes in Candidatus Liberibacter asiaticus during the transmission in distinct hosts between plant and insect. Mol Plant Pathol 14(4):391–404CrossRefGoogle Scholar
  51. 51.
    Ghanim M, Achor D, Ghosh S, Kontsedalov S, Lebedev G, Levy A (2017) ‘Candidatus Liberibacter asiaticus’ accumulates inside endoplasmic reticulum associated vacuoles in the gut cells of Diaphorina citri. Sci Rep 7(1):16945CrossRefGoogle Scholar
  52. 52.
    Cirimotich CM, Dong Y, Clayton AM, Sandiford SL, Souza-Neto JA, Mulenga M, Dimopoulos G (2011) Natural microbe-mediated refractoriness to Plasmodium infection in Anopheles gambiae. Science 332(6031):855–858CrossRefGoogle Scholar
  53. 53.
    Pitino M, Armstrong CM, Duan Y (2017) Molecular mechanisms behind the accumulation of ATP and H2O2 in citrus plants in response to ‘Candidatus Liberibacter asiaticus’ infection. Hortic Res 4:17040CrossRefGoogle Scholar
  54. 54.
    Ren S-L, Li Y-H, Ou D, Guo Y-J, Qureshi JA, Stansly PA, Qiu B-L (2018) Localization and dynamics of infection in Asian citrus psyllid, the insect vector of the causal pathogens of Huanglongbing. Microbiology Open 7(3):e00561CrossRefGoogle Scholar
  55. 55.
    Cicero J et al (2009) The digestive system of Diaphorina citri and Bactericera cockerelli (Hemiptera: Psyllidae). Ann Entomol Soc Am 102(4):650–665CrossRefGoogle Scholar
  56. 56.
    Kruse A, Fattah-Hosseini S, Saha S, Johnson R, Warwick ER, Sturgeon K, Mueller L, MacCoss MJ, Shatters RG, Cilia Heck M (2017) Combining 'omics and microscopy to visualize interactions between the Asian citrus psyllid vector and the Huanglongbing pathogen Candidatus Liberibacter asiaticus in the insect gut. PLoS One 12(6):e0179531CrossRefGoogle Scholar
  57. 57.
    Ramsey J et al (2017) Protein interaction networks at the host–microbe interface in Diaphorina citri, the insect vector of the citrus greening pathogen. R Soc Open Sci 4(2):160545CrossRefGoogle Scholar
  58. 58.
    Killiny N, Nehela Y (2017) Metabolomic response to huanglongbing: role of carboxylic compounds in Citrus sinensis response to 'Candidatus Liberibacter asiaticus' and its vector, Diaphorina citri. Mol Plant-Microbe Interact 30(8):666–678CrossRefGoogle Scholar
  59. 59.
    Ingwell LL, Eigenbrode SD, Bosque-Pérez NA (2012) Plant viruses alter insect behavior to enhance their spread. Sci Rep 2:578CrossRefGoogle Scholar
  60. 60.
    Mann RS, Ali JG, Hermann SL, Tiwari S, Pelz-Stelinski KS, Alborn HT, Stelinski LL (2012) Induced release of a plant-defense volatile ‘deceptively’ attracts insect vectors to plants infected with a bacterial pathogen. PLoS Pathog 8(3):e1002610CrossRefGoogle Scholar
  61. 61.
    Martini X, Hoffmann M, Coy MR, Stelinski LL, Pelz-Stelinski KS (2015) Infection of an insect vector with a bacterial plant pathogen increases its propensity for dispersal. PLoS One 10(6):e0129373CrossRefGoogle Scholar
  62. 62.
    Rohrscheib CE, Bondy E, Josh P, Riegler M, Eyles D, van Swinderen B, Weible II MW, Brownlie JC (2015) Wolbachia influences the production of octopamine and affects Drosophila male aggression. Appl Environ Microbiol 81(14):4573–4580CrossRefGoogle Scholar
  63. 63.
    Dan H, Ikeda N, Fujikami M, Nakabachi A (2017) Behavior of bacteriome symbionts during transovarial transmission and development of the Asian citrus psyllid. PLoS One 12(12):e0189779CrossRefGoogle Scholar
  64. 64.
    Ukuda-Hosokawa R, Sadoyama Y, Kishaba M, Kuriwada T, Anbutsu H, Fukatsu T (2015) Infection density dynamics of the citrus greening bacterium “Candidatus Liberibacter asiaticus” in field populations of the psyllid Diaphorina citri and its relevance to the efficiency of pathogen transmission to citrus plants. Appl Environ Microbiol 81(11):3728–3736CrossRefGoogle Scholar
  65. 65.
    Lopez-Sanchez MJ et al (2008) Blattabacteria, the endosymbionts of cockroaches, have small genome sizes and high genome copy numbers. Environ Microbiol 10(12):3417–3422CrossRefGoogle Scholar
  66. 66.
    Komaki K, Ishikawa H (2000) Genomic copy number of intracellular bacterial symbionts of aphids varies in response to developmental stage and morph of their host. Insect Biochem Mol Biol 30(3):253–258CrossRefGoogle Scholar

Copyright information

© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2018

Authors and Affiliations

  1. 1.Department of Plant Pathology, Faculty of AgricultureTarbiat Modares UniversityTehranIran
  2. 2.Boyce Thompson InstituteIthacaUSA
  3. 3.Plant Pathology and Plant Microbe Biology Section, School of Integrative Plant ScienceCornell UniversityIthacaUSA
  4. 4.Plant Protection Research Department, Hormozgan Agricultural and Natural Resources Research and Education CenterAgricultural Research Education and Extension Organization (AREEO)Bandar AbbasIran
  5. 5.Department of Entomology, Faculty of AgricultureTarbiat Modares UniversityTehranIran
  6. 6.USDA ARS Emerging Pests and Pathogens Research UnitRobert W. Holley Center for Agriculture and HealthIthacaUSA

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