Characterization of the bacterial community in Haemaphysalis longicornis (Acari: Ixodidae) throughout developmental stages

  • Zhang RuilingEmail author
  • Huang Zhendong
  • Yu Guangfu
  • Zhang ZhongEmail author


As one of the most important vectors, Haemaphysalis longicornis can transmit a variety of pathogens and is widely distributed in China. It has been reported that the bacterial community in ticks can impact tick fitness, development, and reproduction and even the transmission of tick-borne pathogens. In this study, bacterial diversity across all developmental stages (eggs, larvae, nymphs and adults) of H. longicornis was investigated using high-throughput sequencing technology. The results demonstrated that Proteobacteria was the dominant phylum and that Coxiella was the most abundant bacterial genus across all the samples. Alpha diversity analysis demonstrated that the eggs had the highest bacterial richness and diversity, and the bacterial community of the larvae was found to be similar to that of the eggs. However, there was a rapid increase in the relative abundance of Coxiella upon development of larvae to nymphs. Females exhibited the lowest bacterial diversity, and the proportion of Coxiella decreased from 85% in females to 45% in males. Our results suggest that H. longicornis lost most of the bacteria present in the early developmental stages and re-established the bacterial community after bloodmeals and molting.


16S rRNA sequencing Proteobacteria Coxiella Tick-borne disease 



This research was supported by development plan project of Shandong province science and technology (No. 2017GSF221017) and National Natural Sciences Foundation of China (No. 81,871,686).

Supplementary material

10493_2019_339_MOESM1_ESM.tif (6 mb)
Rarefaction curves of all samples used in this study (TIF 6096 KB)
10493_2019_339_MOESM2_ESM.tif (23.9 mb)
Most abundant bacterial genus detected in different samples (TIF 24450 KB)


  1. Afzelius BA, Alberti G, Dallai R, Godula J, Witalinski W (1989) Virus-and rickettsia-infected sperm cells in arthropods. J Invertebr Path 53:365–377CrossRefGoogle Scholar
  2. Brackney DE, Armstrong PM (2016) Transmission and evolution of tick-borne viruses. Curr Opin Virol 21:67–74. CrossRefGoogle Scholar
  3. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336. CrossRefGoogle Scholar
  4. Carpi G, Cagnacci F, Wittekindt NE, Zhao F, Qi J, Tomsho LP, Drautz D, Rizzoli A, Schuster SC (2011) Metagenomic profile of the bacterial communities associated with Ixodes ricinus ticks. PLoS One 6:e25604. CrossRefGoogle Scholar
  5. Clay K, Klyachko O, Grindle N, Civitello D, Oleske D, Fuqua C (2008) Microbial communities and interactions in the lone star tick, Amblyomma americanum. Mol Ecol 17:4371–4381CrossRefGoogle Scholar
  6. de la Fuente J, Blouin EF, Kocan KM (2003) Infection exclusion of the rickettsial pathogen Anaplasma marginale in the tick vector Dermacentor variabilis. Clin Diagn Lab Immun 10:182–184. Google Scholar
  7. Duan DY, Cheng TY (2017) Determination of the microbial community features of Haemaphysalis flava in different developmental stages by high-throughput sequencing. J Basic Microbiol 57(4):302–308. CrossRefGoogle Scholar
  8. Epis S, Mandrioli M, Genchi M, Montagna M, Sacchi L, Pistone D, Sassera D (2013) Localization of the bacterial symbiont Candidatus Midichloria mitochondtrii within the hard tick Ixodes ricinus by wholemount FISH staining. Ticks Tick Borne Dis 4:39–45. CrossRefGoogle Scholar
  9. Gall CA, Reif KE, Scoles GA, Mason KL, Mousel M, Noh SM, Brayton KA (2016) The bacterial microbiome of Dermacentor andersoni ticks influences pathogen susceptibility. ISME J 10:1846–1855. CrossRefGoogle Scholar
  10. Gerhart JG, Moses AS, Raghavan R (2016) A Francisella-like endosymbiont in the Gulf Coast tick evolved from a mammalian pathogen. Sci Rep 6:33670. CrossRefGoogle Scholar
  11. Gottlieb Y, Lalzar I, Klasson L (2015) Distinctive genome reduction rates revealed by genomic analyses of two Coxiella-like endosymbionts in ticks. Genome Biol Evol 7:1779–1796. CrossRefGoogle Scholar
  12. Hawlena H, Rynkiewicz E, Toh E, Alfred A, Durden LA, Hastriter MW, Nelson DE, Rong R, Munro D, Dong Q, Fuqua C, Clay K (2013) The arthropod, but not the vertebrate host or its environment, dictates bacterial community composition of fleas and ticks. ISME J 7:221–223. CrossRefGoogle Scholar
  13. Heath A (2016) Biology, ecology and distribution of the tick, Haemaphysalis longicornis Neumann (Acari: Ixodidae) in New Zealand. N Z Vet J 64(1):10–20. CrossRefGoogle Scholar
  14. Heise SR, Elshahed MS, Little SE (2010) Bacterial diversity in Amblyomma americanum (Acari: Ixodidae) with a focus on members of the genus Rickettsia. J Med Entomol 47:258–268CrossRefGoogle Scholar
  15. Hunter DJ, Torkelson JL, Bodnar J, Mortazavi B, Laurent T, Deason J, Thephavongsa K, Zhong J (2015) The Rickettsia endosymbiont of Ixodes pacificus contains all the genes of De novo folate biosynthesis. Plos One 10:e0144552. CrossRefGoogle Scholar
  16. Ixodoidea (1968) Review of Haemaphysalis (Kaiseriana) longicornis Neumann (resurrected) of Australia, New Zealand, New Caledonia, Fiji, Japan, Korea, and Northeastern China and USSR, and its parthenogenetic and bisexual populations. Ixodida) J Parasitol 54(6):1197–1213CrossRefGoogle Scholar
  17. Jongejan F, Uilenber G (2004) The global importance of ticks. Parasitology 129(Suppl):S3–S14. Google Scholar
  18. Kageyama D, Narita S, Watanabe M (2012) Insect sex determination manipulated by their endosymbionts: incidences, mechanisms and implications. Insects 3(1):161–199. CrossRefGoogle Scholar
  19. Kim KH, Yi J, Kim G, Choi SJ, Jun KI, Kim NH, Choe PG, Kim NJ, Lee JK, Oh MD (2013) Severe fever with thrombocytopenia syndrome, South Korea, 2012. Emerg Infect Dis 19:1892–1894. CrossRefGoogle Scholar
  20. Klyachko O, Stein BD, Grindle N, Clay K, Fuqua C (2007) Localization and visualization of a Coxiella-Type symbiont within the lone star tick, Amblyomma americanum. Appl Environ Microbiol 73:6584–6594. CrossRefGoogle Scholar
  21. Liu LM, Liu JN, Liu Z, Yu ZJ, Xu SQ, Yang XH, Tuo L, Li SS, Guo LD, Liu JZ (2013) Microbial communities and symbionts in the hard tick Haemaphysalis longicornis (Acari: Ixodidae) from north China. Parasit Vectors 6:310. CrossRefGoogle Scholar
  22. Liu Q, He B, Huang SY, Wei F, Zhu XQ (2014) Severe fever with thrombocytopenia syndrome, an emerging tick-borne zoonosis. Lancet Infect Dis 14:763–772. CrossRefGoogle Scholar
  23. Lu BL, Wu HY (2003) Classification and Identification of important medical insects of China. Henan Science and technology publishing House. pp661–665 (in Chinese) Google Scholar
  24. Macaluso KR, Sonenshine DE, Ceraul SM, Azad AF (2002) Rickettsial infection in Dermacentor variabilis (Acari: Ixodidae) inhibits transovarial transmission of a second Rickettsia. J Med Entomol 39:809–813CrossRefGoogle Scholar
  25. Machado-Ferreira E, Dietrich G, Hojgaard A, Levin M, Piesman J, Zeidner NS, Soares CA (2011) Coxiella symbionts in the Cayenne tick Amblyomma cajennense. Microb Ecol 62:134–142. CrossRefGoogle Scholar
  26. Mahara F (1997) Japanese spotted fever: report of 31 cases and review of the literature. Emerg Infect Dis 3:105–111CrossRefGoogle Scholar
  27. Matsuo T, Okura N, Kakuda H, Yano Y (2013) Reproduction in a Metastriata tick, Haemaphysalis longicornis (Acari: Ixodidae). J Acarol Soc Japan 22:1–23. CrossRefGoogle Scholar
  28. Menchaca AC, Visi DK, Strey OF, Teel PD, Kalinowski K, Allen MS, Williamson PC (2013) Preliminary assessment of microbiome changes following blood-feeding and survivorship in the Amblyomma americanum nymph-to-adult transition using semiconductor sequencing. PLoS One 8:e67129. CrossRefGoogle Scholar
  29. Narasimhan S, Rajeevan N, Liu L, Zhao YO, Heisig J, Pan J, Eppler-Epstein R, Deponte K, Fish D, Fikrig E (2014) Gut microbiota of the tick vector Ixodes scapularis modulate colonization of the Lyme disease spirochete. Cell Host Microbe 15:58–71. CrossRefGoogle Scholar
  30. Parola P, Raoult D (2001) Ticks and tick borne bacterial diseases in humans: an emerging infectious threat. Clin Inf Dis 32:897–928. CrossRefGoogle Scholar
  31. R Core Team (2014) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL
  32. Rees H (2004) Hormonal control of tick development and reproduction. Parasitology 129:S127–S143CrossRefGoogle Scholar
  33. Roy BC, Estrada-Peña A, Krücken J, Rehman A, Nijhof AM (2018) Morphological and phylogenetic analyses of Rhipicephalus microplus ticks from Bangladesh, Pakistan and Myanmar. Ticks Tick Borne Dis 9(5):1069–1079. CrossRefGoogle Scholar
  34. Rynkiewicz EC, Hemmerich C, Rusch DB, Fuqua C, Clay K (2015) Concordance of bacterial communities of two tick species and blood of their shared rodent host. Mol Ecol 24:2566–2579. CrossRefGoogle Scholar
  35. Segata N, Izard J, Waldron L, Gevers D, Miropolsky L, Garrett WS, Huttenhower C (2011) Metagenomic biomarker discovery and explanation. Genome Biol 12:
  36. Sjödin A, Svensson K, Ohrman C, Ahlinder J, Lindgren P, Duodu S, Johansson A, Colquhoun DJ, Larsson P, Forsman M (2012) Genome characterisation of the genus Francisella reveals insight into similar evolutionary paths in pathogens of mammals and fish. BMC Genom 13:268. CrossRefGoogle Scholar
  37. Smith TA, Driscoll T, Gillespie JJ, Raghavan R (2015) A Coxiella-like Endosymbiont is a potential vitamin source for the Lone Star Tick. Genome Biol Evol 7:831–838. CrossRefGoogle Scholar
  38. Swei A, Kwan JY (2017) Tick microbiome and pathogen acquisition altered by host blood meal. ISME J 11:813–816. CrossRefGoogle Scholar
  39. Takahashi T, Maeda K, Suzuki T, Ishido A, Shigeoka T, Tominaga T, Kamei T, Honda M, Ninomiya D, Sakai T, Senba T, Kaneyuki S, Sakaguchi S, Satoh A, Hosokawa T, Kawabe Y, Kurihara S, Izumikawa K, Kohno S, Azuma T, Suemori K, Yasukawa M, Mizutani T, Omatsu T, Katayama Y, Miyahara M, Ijuin M, Doi K, Okuda M, Umeki K, Saito T, Fukushima K, Nakajima K, Yoshikawa T, Tani H, Fukushi S, Fukuma A, Ogata M, Shimojima M, Nakajima N, Nagata N, Katano H, Fukumoto H, Sato Y, Hasegawa H, Yamagishi T, Oishi K, Kurane I, Morikawa S, Saijo M (2014) The first identification and retrospective study of severe fever with thrombocytopenia syndrome in Japan. J Infect Dis 209:816–827. CrossRefGoogle Scholar
  40. van Overbeek L, Gassner F, van der Plas CL, Kastelein P, Nunes-da Rocha U, Takken W (2008) Diversity of Ixodes ricinus tick-associated bacterial communities from different forests. FEMS Microbiol Ecol 66:2–84. Google Scholar
  41. Van Treuren W, Ponnusamy L, Brinkerhoff RJ, Gonzalez A, Parobek CM, Juliano JJ, Andreadis TG, Falco RC, Ziegler LB, Hathaway N, Keeler C, Emch M, Bailey JA, Roe RM, Apperson CS, Knight R, Meshnick SR (2015) Variation in the microbiota of Ixodes ticks with regard to geography, species, and sex. Appl Environ Microbiol 81:6200–6209. CrossRefGoogle Scholar
  42. Vayssier-Taussat M, Kazimirova M, Hubalek Z, Hornok S, Farkas R, Cosson JF, Bonnet S, Vourch G, Gasqui P, Mihalca AD, Plantard O, Silaghi C, Cutler S, Rizzoli A (2015) Emerging horizons for tick-borne pathogens: from the “one pathogen-one disease” vision to the pathobiome paradigm. Future Microbiol 10:2033–2043. CrossRefGoogle Scholar
  43. 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 7:5261–5267. CrossRefGoogle Scholar
  44. Williamsnewkirk AJ, Rowe LA, Mixsonhayden TR, Dasch GA (2014) Characterization of the bacterial communities of life stages of free living lone star ticks (Amblyomma americanum). Plos One 9(7):e102130. CrossRefGoogle Scholar
  45. Yu XJ, Liang MF, Zhang SY, Liu Y, Li JD, Sun YL, Zhang L, Zhang QF, Popov VL, Li C, Qu J, Li Q, Zhang YP, Hai R, Wu W, Wang Q, Zhan FX, Wang XJ, Kan B, Wang SW, Wan KL, Jing HQ, Lu JX, Yin WW, Zhou H, Guan XH, Liu JF, Bi ZQ, Liu GH, Ren J, Wang H, Zhao Z, Song JD, He JR, Wan T, Zhang JS, Fu XP, Sun LN, Dong XP, Feng ZJ, Yang WZ, Hong T, Zhang Y, Walker DH, Wang Y, Li DX (2011) Fever with thrombocytopenia associated with a novel bunyavirus in China. N Engl J Med 364:1523–1532. CrossRefGoogle Scholar
  46. Yu ZJ, Wang RR, Li NX, Zhang CM, Liu JZ (2017) Microbial diversity of the Tibetan tick Haemaphysalis tibetensis (Acari: Ixodidae). Exp Appl Acarol 73:237–244. CrossRefGoogle Scholar
  47. Zhan J, Wang Q, Cheng J, Hu B, Li J, Zhan F, Song Y, Guo D (2017) Current status of severe fever with thrombocytopenia syndrome in China. Virol Sin 32:51–62. CrossRefGoogle Scholar
  48. Zhang XC, Yang ZN, Lu B, Ma XF, Zhang CX, Xu HJ (2014) The composition and transmission of microbiome in hard tick, Ixodes persulcatus, during blood meal. Ticks Tick Borne Dis 5:864–870. CrossRefGoogle Scholar
  49. Zhong J, Jasinskas A, Barbour AG (2007) Antibiotic treatment of the tick vector Amblyomma americanum reduced reproductive fitness. PLoS One 2:e405. CrossRefGoogle Scholar
  50. Zolnik CP, Prill RJ, Falco RC, Daniels TJ, Kolokotronis SO (2016) Microbiome changes through ontogeny of a tick pathogen vector. Mol Ecol 25:4963–4977CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Collaborative Innovation Center for the Origin and Control of Emerging Infectious DiseasesTaishan Medical UniversityTaianChina
  2. 2.School of Basic Medical ScienceTaishan Medical UniversityTaianChina

Personalised recommendations