Advertisement

Target-Enriched Endosymbiont Sequencing (TEEseq): A New High-Throughput Sequencing Approach Applied to the Comprehensive Characterization of Endosymbionts

  • Hannes SchulerEmail author
  • Jacqueline A. Lopez
  • Meredith M. Doellman
  • Glen R. Hood
  • Scott P. Egan
  • Jeffrey L. Feder
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1858)

Abstract

Intracellular bacteria are ubiquitous in the insect world, with perhaps the best-studied example being the alphaproteobacterium, Wolbachia. Like most endosymbionts, Wolbachia cannot be cultivated outside of its host cells, hindering traditional microbial characterization techniques. Furthermore, multiple Wolbachia strains can be present within a single host, and certain strains can be present in densities below the detection limit of current methods. To date, Wolbachia has most commonly been studied using polymerase chain reaction (PCR) amplification and Sanger DNA sequencing by targeting specific genes in the bacterium’s genome. PCR amplification and Sanger sequencing of multiple Wolbachia strains requires analysis of individually cloned sequences, which is resource and labor intensive. To help mitigate these difficulties, we present a modified double digest restriction site associated DNA sequencing (ddRADseq) approach to target and sequence in parallel multiple genes by adding restriction enzyme recognition sites to gene-specific PCR primers. Adopting this strategy allows us to uniquely tag and sequence amplicons from multiple hosts simultaneously on an Illumina MiSeq platform. Our approach represents an efficient and cost-effective method to characterize multiple target genes in population surveys.

Key words

Wolbachia Endosymbiont Microbial community Metabarcoding PCR enrichment ddRAD Illumina sequencing 

Notes

Acknowledgment

We thank Melissa Stephens, Michael Pfrender, and Stuart Jones for helpful discussions. HS is supported by the Erwin Schrödinger Fellowship J-3527-B22 of the Austrian Science Fund FWF. In addition, the work was aided by support to JLF from the National Science Foundation and United States Department of Agriculture.

Supplementary material

436248_1_En_14_MOESM1_ESM.xlsx (54 kb)
Supplemental Table 1 (XLSX 53 kb)

References

  1. 1.
    Hurst GDD, Frost CL (2015) Reproductive parasitism: maternally inherited symbionts in a biparental world. Cold Spring Harb Perspect Biol 7:a017699CrossRefGoogle Scholar
  2. 2.
    Engelstädter J, Hurst GDD (2009) The ecology and evolution of microbes that manipulate host reproduction. Annu Rev Ecol Evol S 40:127–149CrossRefGoogle Scholar
  3. 3.
    Weinert LA, Araujo-Jnr EV, Ahmed MZ, Welch JJ (2015) The incidence of bacterial endosymbionts in terrestrial arthropods. Proc R Soc B 282:20150249CrossRefGoogle Scholar
  4. 4.
    Wu M, Sun LV, Vamathevan J et al (2004) Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS Biol 2:327–341CrossRefGoogle Scholar
  5. 5.
    Klasson L, Westberg J, Sapountzis P et al (2009) The mosaic genome structure of the Wolbachia wRi strain infecting Drosophila simulans. Proc Nat Acad Sci U S A 106:5725–5730CrossRefGoogle Scholar
  6. 6.
    Ellegaard KM, Klasson L, Näslund K et al (2013) Comparative genomics of Wolbachia and the bacterial species concept. PLoS Genet 9:e1003381CrossRefGoogle Scholar
  7. 7.
    Braig HR, Zhou WG, Dobson SL, O'Neill SL (1998) Cloning and characterization of a gene encoding the major surface protein of the bacterial endosymbiont Wolbachia pipientis. J Bacteriol 180:2373–2378PubMedPubMedCentralGoogle Scholar
  8. 8.
    Baldo L, Dunning Hotopp JC, Jolley KA et al (2006) Multilocus sequence typing system for the endosymbiont Wolbachia pipientis. Appl Environ Microbiol 72:7098–7110CrossRefGoogle Scholar
  9. 9.
    Baldo L, Ayoub NA, Hayashi CY et al (2008) Insight into the routes of Wolbachia invasion: high levels of horizontal transfer in the spider genus Agelenopsis revealed by Wolbachia strain and mitochondrial DNA diversity. Mol Ecol 17:557–569CrossRefGoogle Scholar
  10. 10.
    Schuler H, Arthofer W, Riegler M et al (2011) Multiple Wolbachia infections in Rhagoletis pomonella. Entomol Exp Appl 139:138–144CrossRefGoogle Scholar
  11. 11.
    Stahlhut JK, Desjardins CA, Clark ME et al (2010) The mushroom habitat as an ecological arena for global exchange of Wolbachia. Mol Ecol 19:1940–1952CrossRefGoogle Scholar
  12. 12.
    Ahmed MZ, Breinholt JW, Kawahara AY (2016) Evidence for common horizontal transmission of Wolbachia among butterflies and moths. BMC Evol Biol 16:118CrossRefGoogle Scholar
  13. 13.
    Schuler H, Bertheau C, Egan SP et al (2013) Evidence for a recent horizontal transmission and spatial spread of Wolbachia from endemic Rhagoletis cerasi (Diptera: Tephritidae) to invasive Rhagoletis cingulata in Europe. Mol Ecol 22:4101–4111CrossRefGoogle Scholar
  14. 14.
    Arthofer W, Riegler M, Schneider D et al (2009) Hidden Wolbachia diversity in field populations of the European cherry fruit fly, Rhagoletis cerasi (Diptera, Tephritidae). Mol Ecol 18:3816–3830CrossRefGoogle Scholar
  15. 15.
    Arthofer W, Riegler M, Avtzis DN, Stauffer C (2009) Evidence for low-titre infections in insect symbiosis: Wolbachia in the bark beetle Pityogenes chalcographus (Coleoptera, Scolytinae). Environ Microbiol 11:1923–1933CrossRefGoogle Scholar
  16. 16.
    Gerth M, Bleidorn C (2016) Comparative genomics provides a timeframe for Wolbachia evolution and exposes a recent biotin synthesis operon transfer. Nat Microbiol 2:16241CrossRefGoogle Scholar
  17. 17.
    Bleidorn C, Gerth M (2018) A critical re-evaluation of multilocus sequence typing (MLST) efforts in Wolbachia. FEMS Microbiol Ecol 94(1), fix163.Google Scholar
  18. 18.
    Parchman T, Gompert Z, Benkman C, Schilkey F, Mudge J, Buerkle CA (2012) Genome wide association mapping of an adaptive trait in lodgepole pine. Mol Ecol 21:2991–3005CrossRefGoogle Scholar
  19. 19.
    Baird NA, Etter PD, Atwood TS et al (2008) Rapid SNP discovery and genetic mapping using sequenced RAD markers. PLoS One 3:e3376CrossRefGoogle Scholar
  20. 20.
    Peterson BK, Weber JN, Kay EH et al (2012) Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species. PLoS One 7:e37135CrossRefGoogle Scholar
  21. 21.
    Schloss PD, Westcott SL, Ryabin T et al (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Hannes Schuler
    • 1
    • 2
    Email author
  • Jacqueline A. Lopez
    • 3
  • Meredith M. Doellman
    • 1
  • Glen R. Hood
    • 1
    • 4
  • Scott P. Egan
    • 5
  • Jeffrey L. Feder
    • 1
  1. 1.Department of Biological Sciences, Galvin Life Sciences BuildingUniversity of Notre DameNotre DameUSA
  2. 2.Faculty of Science and TechnologyFree University of Bozen-BolzanoBolzanoItaly
  3. 3.Genomics and Bioinformatics Core FacilityUniversity of Notre DameNotre DameUSA
  4. 4.Department of Biological SciencesWayne State UniversityDetroitUSA
  5. 5.Department of BioSciencesRice UniversityHoustonUSA

Personalised recommendations