Parasitology Research

, Volume 116, Issue 7, pp 1899–1906 | Cite as

Bacterial diversity of cosmopolitan Culex pipiens and invasive Aedes japonicus from Germany

  • Sina ZotzmannEmail author
  • Antje Steinbrink
  • Kathrin Schleich
  • Felix Frantzmann
  • Chinhda Xoumpholphakdy
  • Manuela Spaeth
  • Claire Valiente Moro
  • Patrick Mavingui
  • Sven Klimpel
Original Paper


Symbiotic bacteria have gained significant attention in recent years. For example, microbiota of some mosquito species seems to influence the development and transmission of pathogens. Furthermore, several attempts using bacteria as a paratransgenetic tool have been made in order to assist the control of mosquito-borne diseases. In this study, we examined the bacterial diversity of wild-caught adult Culex (Cx.) pipiens and laboratory-reared adult Aedes japonicus (Ae. japonicus) in Germany using a culture-independent method. Genomic DNA was extracted from each specimen and submitted to PCR amplification of eubacterial 16S rDNA. After the cloning reaction, 28 bacterial transformants per sample containing the 16S rDNA inserts were selected per each sample for sequencing. The analysed specimens of Cx. pipiens as well as of Ae. japonicus showed a diverse bacterial community including some common bacterial genera. Blast analysis allowed to identify 21 bacterial genera belonging to 2 phyla among the 23 specimens of Cx. pipiens. The 14 analysed Ae. japonicus revealed 11 bacterial genera belonging to 3 phyla. In both mosquito species, identified isolates were mainly Proteobacteria. Only 4 of the bacterial genera were found in both mosquito species, with the most prevalent genera Sphingomonas and Rahnella in Cx. pipiens and in Ae. japonicus respectively. Most of the bacterial genera found in our study have been identified in other mosquito species before. Due to the currently scarce data situation, ongoing examinations on the very abundant bacterial genera or species are strongly required to determine their relevance for the biology and adaptiveness of mosquitoes including pathogen-host relationship.


Bacterial diversity Culex pipiens Aedes japonicus Cloning 



This present research was funded by the ERA-Net BiodivERsA, with the national funders DFG KL 2087/6-1, FWF I-1437 and ANR-13-EBID-0007-01 as part of the 2012-13 BiodivERsA call for research proposals and by the German Federal Ministry of Food and Agriculture (BMEL) through the Federal Office for Agriculture and Food (BLE), grant number 2819105115 as well as by the Uniscientia Foundation (P 121-2017). We thank Birgit Nagel and Gabriele Elter for their technical assistance in the molecular laboratory. We also thank Dr. Rüdiger Berghahn and Ronny Schmiediche from the German Federal Environmental Agency (UBA) in Berlin for mosquito sampling.


  1. 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 25:3389–3402CrossRefPubMedPubMedCentralGoogle Scholar
  2. Becker N, Petrić D, Zgomba M, Boase C, Dahl C, Madon M, Kaiser A (2010) Mosquitoes and their control, 2nd edn. Springer-Verlag, Berlin HeidelbergCrossRefGoogle Scholar
  3. Becker N, Huber K, Pluskota B, Kaiser A (2011) Ochlerotatus japonicus japonicus—a newly established neozoan in Germany and a revised list of the German mosquito fauna. Eur Mosq Bull 29:88–102Google Scholar
  4. Becker N, Krüger A, Kuhn C, Plenge-Bönig A, Thomas SM, Schmidt-Chanasit J, Tannich E (2014) Stechmücken als Überträger exotischer Krankheitserreger in Deutschland. Bundesgesundheitsblt 57:531–540. doi: 10.1007/s00103-013-1918-8 CrossRefGoogle Scholar
  5. Berg RD (1996) The indigenous gastrointestinal microflora. Trends Microbiol 11:430–435CrossRefGoogle Scholar
  6. Bonilauri P, Bellini R, Calzolari M, Angelini R, Venturi L, Fallacara F, Cordioli P, Angelini P, Venturelli C, Merialdi G, Dottori M (2008) Chikungunya virus in Aedes albopictus, Italy. Emerg Infect Dis 14:852–854. doi: 10.3201/eid1405.071144 CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cancrini G, Scaramozzino P, Gabrielli S, Di Paolo M, Toma L, Romi R (2007) Aedes albopictus and Culex pipiens implicated as natural vectors of Dirofilaria repens in Central Italy. J Med Entomol 44:1064–1066PubMedGoogle Scholar
  8. Caraballo H, King K (2014) Emergency department management of mosquito-borne illness: malaria, dengue, and West Nile virus. Emerg Med Pract 16:1–23PubMedGoogle Scholar
  9. Charan SS, Pawar KD, Severson DW, Patole MS, Shouche YS (2013) Comparative analysis of midgut bacterial communities of Aedes aegypti mosquito strains varying in vector competence to dengue virus. Parasitol Res 112:2627–2637. doi: 10.1007/s00436-013-3428-x CrossRefPubMedGoogle Scholar
  10. Coutinho-Abreu IV, Zhu KY, Ramalho-Ortigao M (2010) Transgenesis and paratransgenesis to control insect-borne diseases: current status and future challenges. Parasitol Int 59:1–8. doi: 10.1016/j.parint.2009.10.002 CrossRefPubMedGoogle Scholar
  11. Demaio J, Pumpuni CB, Kent M, Beier JC (1996) The midgut bacterial flora of wild Aedes triseriatus, Culex pipiens, and Psorophora columbiae mosquitoes. AmJTrop Med Hyg 54:219–223CrossRefGoogle Scholar
  12. De Vos P, Van Landschoot A, Segers P, Tytgat R, Gillis M, Bauwens M, Rossau R, Goor M, Pot B, Kersters K, Lizzaraga P, De Ley J (1989) Genotypic relationships and taxonomic localization of unclassified Pseudomonas and Pseudomonas-like strains by deoxyribonucleic acid: ribosomal ribonucleic acid hybridizations. Int J Syst Bacteriol 39:35–49CrossRefGoogle Scholar
  13. Dhayal D, Parasher H, Sharma A, Kumar P, Adak T, Jaiwal R (2014) Diversity of culturable midgut bacteria of Indian malarial vector Anopheles stephensi. IJARM 2:305–311Google Scholar
  14. Diaz-Badillo A, Bolling BG, Perez-Ramirez G, Moore CG, Martinez-Munoz JP, Padilla-Viveros AA, Camacho-Nuez M, Diaz-Perez A, Beaty BJ, Lourdes Munoz M (2011) The distribution of potential West Nile virus vectors, Culex pipiens pipiens and Culex pipiens quinquefasciatus (Diptera: Culicidae), in Mexico City. Parasite Vectors 4:70. doi: 10.1186/1756-3305-4-70 CrossRefGoogle Scholar
  15. Dillon RJ, Vennard CT, Buckling A, Charnley AK (2005) Diversity of locust gut bacteria protects against pathogen invasion. Ecol Lett 8:1291–1298. doi: 10.1111/j.1461-0248.2005.00828.x CrossRefGoogle Scholar
  16. Dinparast Djadid N, Jazayeri H, Raz A, Favia G, Ricci I, Zakeri S (2011) Identification of the midgut microbiota of An. stephensi and An. maculipennis for their application as a paratransgenetic tool against malaria. PLoS One 6:e28484. doi: 10.1371/journal.pone.0028484
  17. Edwards U, Rogall T, Blöcker H, Emde M, Böttger EC (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17:7843–7853CrossRefPubMedPubMedCentralGoogle Scholar
  18. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit 1 from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3(5):294–299PubMedGoogle Scholar
  19. Fonseca DM, Keyghobadi N, Malcolm CA, Mehmet C, Schaffner F, Mogi M, Fleischer RC, Wilkerson RC (2004) Emerging vectors in the Culex pipiens complex. Science 303:1535–1538. doi: 10.1126/science.1094247 CrossRefPubMedGoogle Scholar
  20. Guerreo R, Margulis L, Berlanga M (2013) Symbiogenesis: the holobiont as a unit of evolution. Int Microbiol 16:133–143. doi: 10.2436/20.1501.01.188 Google Scholar
  21. Guo Y, Jiao Z, Li L, Wu D, Crowley DE, Wang Y, Wu W (2012) Draft genome sequence of Rahnella aquatilis strain HX2, a plant growth-promoting Rhizobacterium isolated from vineyard soil in Beijing, China. J Bacteriol 194:6646–6647. doi: 10.1128/JB.01769-12 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Gutsevich AV, Monchadskii AS, Shtakel’berg AA (1974) Fauna of the U.S.S.R. NS: Diptera: mosquitoes: family Culicidae. 2nd ed. Leningrad. Akademiya Nauk SSSR-Zoologicheskii institute, LeningradGoogle Scholar
  23. Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser 41:95–98Google Scholar
  24. Hedges LM, Brownlie JC, O’Neill SL, Johnson KN (2008) Wolbachia and virus protection in insects. Science 322:702. doi: 10.1126/science.1162418 CrossRefPubMedGoogle Scholar
  25. Hesson JC, Rettich F, Merdić E, Vignjević G, Östman Ö, Schäfer M, Schaffner F, Foussadier R, Besnard G, Medlock J, Scholte EJ, Lundström JO (2014) The arbovirus vector Culex torrentium is more prevalent than Culex pipiens in northern and central Europe. Med Vet Entomol 28:179–186. doi: 10.1111/mve.12024 CrossRefPubMedGoogle Scholar
  26. Heumann W (1960) Versuche zur Rekombination sternbildender Bakterien. Naturwissenschaften 47:330–331CrossRefGoogle Scholar
  27. Hongoh Y, Deevong P, Inoue T, Moriya S, Trakulnaleamsai S, Ohkuma M, Vongkaluang C, Noparatnaraporn N, Kudo T (2005) Intra- and interspecific comparison of bacterial diversity and community structure support coevolution of gut microbiota and termite host. Appl Environ Microb 71:6590–6599. doi: 10.1128/AEM.71.11.6590-6599.2005 CrossRefGoogle Scholar
  28. Hugenholtz P, Goebel BM, Pace NR (1998) Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J Bacteriol 180:4765–4774PubMedPubMedCentralGoogle Scholar
  29. Jöst H, Bialonski A, Storch V, Günther S, Becker N, Schmidt-Chanasit J (2010) Isolation and phylogenetic analysis of Sindbis viruses from mosquitoes in Germany. J Clin Microbiol 48:1900–1903. doi: 10.1128/JCM.00037-10 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Jöst H, Bialonski A, Maus D, Sambri V, Eiden M, Groschup MH, Günther S, Becker N, Schmidt-Chanasit J (2011) Isolation of Usutu virus in Germany. AmJTrop Med Hyg 85:551–553. doi: 10.4269/ajtmh.2011.11-0248 CrossRefGoogle Scholar
  31. Kageyama D, Narita S, Watanabe M (2012) Insect sex determination manipulated by their endosymbionts: incidences, mechanisms and implications. Insects 3:161–199. doi: 10.3390/insects3010161 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Kambris Z, Cook PE, Phuc HK, Sinkins SP (2009) Immune activation by life-shortening Wolbachia and reduced filarial competence in mosquitoes. Science 326:134–136. doi: 10.1126/science.1177531 CrossRefPubMedPubMedCentralGoogle Scholar
  33. Kim C-H, Lampman R, Muturi EJ (2015) Bacterial communities and midgut microbiota associated with mosquito population from waste tires in east-central Illinois. J Med Entomol 52:63–75. doi: 10.1093/jme/tju011 CrossRefPubMedGoogle Scholar
  34. Kim HS, Lee OK, Lee SJ, Hwang S, Kim SJ, Yang SH, Park S, Lee EY (2006) Enantioselective epoxide hydrolase activity of a newly isolated microorganism, Sphingomonas echinoides EH-983, from seawater. J Mol Catal B-Enzym 41:130–135. doi: 10.1016/j.molcatb.2006.05.009 CrossRefGoogle Scholar
  35. Koch H, Schmid-Hempel P (2011) Socially transmitted gut microbiota protect bumble bees against an intestinal parasite. P Natl Acad Sci 108:19288–19292. doi: 10.1073/pnas.111047410 CrossRefGoogle Scholar
  36. Köhler T, Dietrich C, Scheffrahn RH, Brune A (2012) High-resolution analysis of gut environment and bacterial microbiota reveals functional compartmentation of the gut in wood-feeding higher termites. Appl Environ Microb 78:4691–4701. doi: 10.1128/AEM.00683-12 CrossRefGoogle Scholar
  37. Kumar NP, Rajavel AR, Natarajan R, Jambulingam P (2007) DNA barcodes can distinguish species of Indian mosquitoes (Diptera: Culicidae). J Med Entomol 44:1–7. doi: 10.1093/jmedent/41.5.01 CrossRefPubMedGoogle Scholar
  38. La Ruche G, Souares Y, Armengaud A, Peloux-Petiot F, Delaunay P, Despres P, Lenglet A, Jourdain F, Leparc-Goffart I, Charlet F, Ollier L, Mantey K, Mollet T, Fournier JP, Torrents R, Leitmeyer K, Hilairet P, Zeller H, Van Bortel W, Dejour-Salamanca D, Grandadam M, Gastellu-Etchegorry M (2010) First two autochthonous dengue virus infections in metropolitan France, September 2010. Euro Surveill 15:19676PubMedGoogle Scholar
  39. Martinez RJ, Bruce D, Detter C, Goodwin LA, Han J, Han CS, Held B, Land ML, Mikhailova N, Nolan M, Pennacchio L, Pitluck S, Tapia R, Woyke T, Sobecky PA (2012) Complete genome sequence of Rahnella aquatilis CIP 78.65. J Bacteriol 194:3020–3021. doi: 10.1128/JB.00380-12 CrossRefPubMedPubMedCentralGoogle Scholar
  40. Melaun C, Werblow A, Cunze S, Zotzmann S, Koch LK, Mehlhorn H, Dörge DD, Huber K, Tackenberg O, Klimpel S (2015) Modeling of the putative distribution of the arbovirus vector Ochlerotatus japonicus japonicus (Diptera: Culicidae) in Germany. Parsitol Res 114:1051–1061. doi: 10.1007/s00436-014-4274-1 CrossRefGoogle Scholar
  41. Minard G, Mavingui P, Moro CV (2013) Diversity and function of bacterial microbiota in the mosquito holobiont. Parasite Vector 6:146. doi: 10.1186/1756-3305-6-146 CrossRefGoogle Scholar
  42. Minard G, Tran FH, van Tran Van CG, Goubert C, Bellet C, Lambert G, Kim KL, Thuy TH, Mavingui P, Moro CV (2015) French invasive Asian tiger mosquito populations harbor reduced bacterial microbiota and genetic diversity compared to Vietnamese autochthonous relatives. Front Microbiol 6:970. doi:  10.3389/fmicb.2015.00970
  43. 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. doi: 10.1603/0022-2585-38.1.29 CrossRefPubMedGoogle Scholar
  44. Moro CV, Tran FH, Raharimalala FN, Ravelonandro P, Mavingui P (2013) Diversity of culturable bacteria including Pantoea in wild mosquito Aedes albopictus. BMC Microbiol 13:70. doi: 10.1186/1471-2180-13-70 CrossRefGoogle Scholar
  45. Mühlhardt C (2009) Der Experimentator Molekularbiologie/Genomics. 6th edn. Spektrum Akademischer Verlag HeidelbergGoogle Scholar
  46. Ngo CT, Aujoulat F, Veas F, Jumas-Bilak E, Manguin S (2015) Bacterial diversity associated with wild caught Anopheles mosquito from Dak Nong province, Vietnam using culture and DNA fingerprint. PLoS One 10:e0118634. doi: 10.1371/journal.pone.0118634 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Osei-Poku J, Mbogo CM, Palmer WJ, Jiggins FM (2012) Deep sequencing reveals extensive variation in the gut microbiota of wild mosquitoes from Kenya. Mol Ecol 21:5138–5150. doi: 10.1111/j.1365-294X.2012.05759.x CrossRefPubMedGoogle Scholar
  48. Pidiyar VJ, Jangid K, Patole MS, Shouche Y (2004) Studies on cultured and uncultured microbiota of wild Culex qiunquefasciatus mosquito midgut based on 16S ribosomal RNA gene analysis. AmJTrop Med Hyg 70:597–603Google Scholar
  49. Ramzy RMR, Farid HA, Kamal IH, Ibrahim GH, Morsy ZS, Faris R, Weil GJ, Williams SA, Gad AM (1997) A polymerase chain reaction-based assay for detection of Wuchereria bancrofti in human blood and Culex pipiens. Trans R Soc Trop Med Hyg 91:156–160CrossRefPubMedGoogle Scholar
  50. Rani A, Sharma A, Rajagopal R, Adak T, Bhatnagar RK (2009) Bacterial diversity analysis of larvae and adult midgut microflora using culture-dependent and culture-independent methods in laboratory-reared and field-collected Anopheles stephensi—an Asian malaria vector. BMC Microbiol 9:96. doi: 10.1186/1471-2180-9-96 CrossRefPubMedPubMedCentralGoogle Scholar
  51. Sardelis MR, Turell MJ (2001) Ochlerotatus j. japonicus in Frederick County, Maryland: discovery, distribution, and vector competence for West Nile virus. J Am Mosq Control Assoc 17:137–141PubMedGoogle Scholar
  52. Schaffner F, Kaufmann C, Hegglin D, Mathis A (2009) The invasive mosquito Aedes japonicus in Central Europe. Med Vet Entomol 23:448–451CrossRefPubMedGoogle Scholar
  53. Schaffner F, Vazeille M, Kaufmann C, Failloux A-B, Mathis A (2011) Vector competence of Aedes japonicus for chikungunya and dengue viruses. Eur Mosq Bull 29:141–142Google Scholar
  54. Tanaka K, Mizusawa K, Saugstad ES (1979) A revision of the adult and larval mosquitoes of Japan (including the Ryukyu Archipelago and the Ogasawara Islands) and Korea (Diptera: Culicidae). Contrib Amer Ent Inst 16:419–422Google Scholar
  55. Wang Y, Gilbreath TM, Kukutla P, Yan G, Xu J (2011) Dynamic gut microbiom across life history of the malaria mosquito Anopheles gambiae in Kenya. PLoS One 6:e24767. doi: 10.1371/journal.pone.0024767 CrossRefPubMedPubMedCentralGoogle Scholar
  56. Werblow A, Klimpel S, Bolius S, Dorresteijn AWC, Sauer J, Melaun C (2014) Population structure and distribution patterns of the sibling mosquito species Culex pipiens and Culex torrentium (Diptera: Culicidae) reveal different evolutionary paths. PLoS One 9:e102158. doi: 10.1371/journal.pone.0102158 CrossRefPubMedPubMedCentralGoogle Scholar
  57. Werren JH (1997) Biology of Wolbachia. Annu Rev Entomol 42:587–609CrossRefPubMedGoogle Scholar
  58. World Health Organization (2016) Vector-borne diseases. Available at: 387/en/ (accessed 04.10.2017)
  59. Yildrim A, Inci A, Duzlu O, Biskin Z, Ica A, Sahin I (2011) Aedes vexans and Culex pipiens as the potential vectors of Dirofilaria immitis in Central Turkey. Vet Parasitol 178:1543–1147Google Scholar
  60. Zouache K, Raharimalala FN, Raquin V, Tran-Van V, Raveloson LHR, Ravelonandro P, Mavingui P (2011) Bacterial diversity of field-caught mosquitoes, Aedes albopictus and Aedes aegypti, from different geographic regions of Madagascar. FEMS Microbiol Ecol 75:377–389. doi: 10.1111/j.1574-6941. 2010.01012.x CrossRefPubMedGoogle Scholar
  61. Zouache K, Michelland RJ, Failloux A-B, Grundmann GL, Mavingui P (2012) Chikungunya virus impacts the diversity of symbiotic bacteria in mosquito vector. Mol Ecol 21:2297–2309. doi: 10.1111/j.1365-294X.2012.05526.x CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Sina Zotzmann
    • 1
    Email author
  • Antje Steinbrink
    • 1
  • Kathrin Schleich
    • 1
  • Felix Frantzmann
    • 1
  • Chinhda Xoumpholphakdy
    • 1
  • Manuela Spaeth
    • 1
  • Claire Valiente Moro
    • 2
  • Patrick Mavingui
    • 2
    • 3
  • Sven Klimpel
    • 1
  1. 1.Institute for Ecology, Evolution and Diversity, Goethe-University (GU); Senckenberg Biodiversity and Climate Research Centre (BiK-F); Senckenberg Gesellschaft für Naturforschung (SGN)Frankfurt/M.Germany
  2. 2.Ecologie MicrobienneUniversité Claude Bernard LyonVilleurbanneFrance
  3. 3.Université de La Réunion, UMR PIMIT, INSERM 1187, CNRS 9192, IRD 249. Plateforme Technologique CYROIRéunionFrance

Personalised recommendations