Zhang etal esa_2015_weevil_bacteria_symbionts

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  • Lech Borowiec

    Diversity and evolution of bacterial symbionts in weevils (Curculionoidea)

    Guanyang Zhang, Patrick Browne, Zhen Geng Hinsby Cadillo-Quiroz & Nico Franz Arizona State University Entomological Society of America 2015 Annual Meeting Minneapolis, Nov 17, 2015

    http://www.taxonbytes.org/

  • The knowns and unknowns of bacterial symbiosis

    in weevils (BACKGROUND)

  • Nowhere else aside from the cicadas do so many symbiotic sites exist as in this insect family [Curculionoidea]

    - P. Buchner (1965, p. 160)

    Diversity of symbiotic sites in weevils

  • Diversity of symbiotic sites in weevils

  • Diversity of weevils (Curculionoidea)

    ~70,000 described species >220,000 estimated

    https://www.flickr.com/photos/budak/https://www.flickr.com/photos/treegrow/https://www.flickr.com/photos/treegrow/https://www.flickr.com/photos/treegrow/

  • Diversity of weevils (Curculionoidea)

    Occupy diverse habitats

    https://klsa.wordpress.com/published-material/milfoil-weevil-guide/https://www.blogger.com/profile/00403062292397104209https://www.blogger.com/profile/00403062292397104209

  • Diversity of weevils (Curculionoidea)

    Phytophagous or fungivorous

  • Diversity of weevils (Curculionoidea)

    h2omanchester.co.uk

    Wood-boring evolved multiple times

    http://h2omanchester.co.uk/faq/woodworm/

  • Discoveries of weevil-specific symbionts

  • Discoveries of weevil-specific symbionts

  • Discoveries of weevil-specific symbionts

  • Weevil-specific bacteria: locations & functions

    Locations Functions

    SOPE Ovaries, bacteriomes

    Nutrient provision, cuticle synthesis

    Nardonella Ovaries, bacteriomes

    Growth & development

    Curculioniphilus Ovaries, bacteriomes

    Unknown

  • Other symbionts in weevils

    Locations Functions

    Spiroplasma ? ?

    Wolbachia ? Parasitoid defense (?)

    Rickettsia ? ?

    Klebsiella Gut, free-living

    Nitrogen fixation

  • Limited taxonomic sampling in previous studies

    Bacteria sequenced?

    Phylogeny of families within the Curculionoidea (Oberprieler et al. 2007)

    Cu

    rcu

    lio

    no

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    a

    - Six non-curculionid weevil families never been sampled

    - Diversity within Curculionidae (true weevils) poor represented; 7/~25 subfamilies sampled

  • Objective: Survey bacterial symbionts in weevils (Superfamily Curculionoidea) using a large taxonomic sample

  • Specimens crushed DNA sequenced Data crunched

    (MATERIALS & METHODS)

  • Specimen & taxonomic sampling

    - 246 weevil and other beetle specimens (88% females, 12% males) dissected and total gut content extracted

    - 124 with usable PCR amplicons (115 weevils, 9 other beetles)

    - 4 families and 17 subfamilies (1 and 7 previously)

    Cu

    rcu

    lio

    no

    ide

    a

  • - Gut content subjected to bead-beating & DNA extracted with Qiagen DNeasy Blood & Tissue kit

    - 16S V3-V5 region amplified with primer pair 515-909 (394 bp); primers barcoded

    - PCR products purified, normalized and sequenced on Illumina MiSeq (paired-end)

    Molecular experiments

  • - Sequences clustered into OTUs (Opertional Taxonomic Units) or Phylotypes in UCLUST at 97% similarity threshold

    - OTUs then queried against Greenegene database for taxonomic assignment, at 97% similarity threshold

    - Diversity of OTUs analyzed and visualizd in QIIME (Quantitative Insights Into Microbial Ecology)

    - Devised reciprocal blast + phylogeny-based method to identify weevil-specific symbiont sequences

    Sequence processing & analyses

  • What symbionts are found in weevils? Total phylogenetic diversity of bacterial symbionts and their occurrence frequencies

  • All bacterial OTUs across samples

    11,464,419 reads

    947 OTUs

  • Total phylogenetic diversity of bacterial symbionts in weevils

    Figure modified from Moran et al. (2008) Annual Review of Genetics, showing phylogenetic distribution of bacterial symbionts

  • Total phylogenetic diversity of bacterial symbionts in weevils

    Figure modified from Moran et al. (2008) Annual Review of Genetics, showing phylogenetic distribution of bacterial symbionts

  • Total phylogenetic diversity of bacterial symbionts in weevils

    Figure modified from Moran et al. (2008) Annual Review of Genetics, showing phylogenetic distribution of bacterial symbionts

  • Total phylogenetic diversity of bacterial symbionts in weevils

    Figure modified from Moran et al. (2008) Annual Review of Genetics, showing phylogenetic distribution of bacterial symbionts

  • >39.1% unassigned or insufficiently identified

    - 11,464,419 sequence reads

    4,482,587 unassigned

  • O symbionts, where art thou? The quest for weevil-specific symbionts (Ca. Nardonella, Ca. Curculioniphilus & SOPE)

  • Reciprocal BLAST

  • Reciprocal BLAST

  • Reciprocal BLAST

  • Reciprocal BLAST

  • Phylogenetic placement of symbiont-suspects

  • Phylogenetic placement of symbiont-suspects

  • Phylogenetic placement of symbiont-suspects

  • Phylogenetic placement of symbiont-suspects

  • Are weevil-specific symbionts conserved in and restricted to weevils? Looking for Americas next top model (symbiont)

  • Phylogeny of Ca. Nardonella & host association

    - Ca. Nardonella is monophyletic - All weevils recovered with this symbiont (n=115)

  • Phylogeny of Ca. Nardonella & host association

    - Ca. Nardonella is monophyletic - All weevils recovered with this symbiont (n=115) - Other beetles (n=9) also with Ca. Nardonella

  • Phylogeny of Ca. Curculioniphilus

    - Ca. Curculioniphilus is monophyletic - Subsampling intensity to be augmented

  • Phylogeny of SOPE & Sodalis allies

    - Sodalis polyphyletic - Three regions: Hemiptera+weevils, tsetse flies-1, tsetse flies-2 - Lacks genetic divergence (1-2%)

  • Conclusion

    - Bacterial symbionts in weevils are diverse

    - Ca. Nardonella is conserved in weevils, but may be more widespread

    - Distance-based OTU/phylotype clustering could not identify symbionts that are highly divergent and poorly represented in existing sequences

    - Reciprocal BLAST + phylogeny is an effective alternative method for identifying symbiont sequences

    - Tree-based method can be limited by the lack of phylogenetic resolution or when monophyly of bacterial groups is not attained.

  • New era of systematic documentation of bacterial symbionts in insects, much of which remain unexplored

  • Acknowledgements - NSF DEB-1155984 (to N. Franz)

    - USDA (Agreement No. 58-1275-1-335; to N. Franz)

    - ASU SOLS Postdoctoral Research Collaborative Grant

    - Andrew Johnston (Reciprocal BLAST)

    - Collaborators: Patrick Browne, Zhen Geng & Hinsby

    Cadillo-Quiroz

    - Franz Lab: Sal Anzaldo, Andrew Jansen & Dr. Sangmi Lee

    http://www.taxonbytes.org/

  • Thanks! Any questions?

    You can find me at: @GYZhang2 GYZ151@Gmail.com Taxonbytes.org Somanyinsects.org

    Slides @ www.slideshareslideshare.net/taxonbytes & ResearchGate

    mailto:GYZ151@Gmail.comhttp://www.slideshareslideshare.net/taxonbyteshttp://www.slideshareslideshare.net/taxonbyteshttp://www.slideshareslideshare.net/taxonbytes