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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)
Cu
rcu
lio
no
ide
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 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 [email protected] Taxonbytes.org Somanyinsects.org
Slides @ www.slideshareslideshare.net/taxonbytes & ResearchGate
