zhang etal esa_2015_weevil_bacteria_symbionts

© 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

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

Diversity of weevils (Curculionoidea)

Occupy diverse habitats

Diversity of weevils (Curculionoidea)

Phytophagous or fungivorous

Diversity of weevils (Curculionoidea)

h2omanchester.co.uk

Wood-boring evolved multiple times

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)

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- 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)

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- 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 America’s 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

Thanks! Any questions?

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

Slides @ www.slideshareslideshare.net/taxonbytes & ResearchGate