Presented at the 2015 meeting of the American Society for Microbiology, May 31, 1045 am-1200 pm

1. Zhen Geng^, Patrick Browne, Guanyang Zhang, Nico Franz, Hinsby Cadillo-Quiroz
School of Life Sciences, ^SOLS Undergraduate Research Program, Arizona State University
Diversity of Bacterial Symbionts in Weevils (Insecta: Curculionoidea)
• 246 specimens of weevils and related beetles were
dissected and their gut contents isolated.
• Samples were subjected to bead-beating with 1 mm and
3 mm-diameter glass beads.
• DNA extraction was performed using Qiagen DNeasy
Blood & Tissue Kit.
• For 110 specimens, a fragment of 16s was amplified
using PCR with primer combination 515F-909R;
primers were barcoded.
• Gel extraction of target fragment was performed with
Promega Quantum PrepTM Freeze ‘N Squeeze DNA
Gel Extraction Kit.
• PCR products were quantified with Nanodrop.
• PCR products were normalized, pooled and sequenced
with Illumina Miseq.
• Sequence identification and diversity analyses were
performed with QIIME.
Materials and Methods
With ~70,000 described species worldwide, weevils (Insecta:
Coleoptera: Curculionoidea) (Fig. 6) are among the most
diverse insect lineages and represent a potential premier
model system for studying bacterial symbiosis. Earlier
traditional methods were based on histological examinations,
which were limited in power and resolution in the detection
of gut-associated bacteria. Molecular detection using the
Polymerase Chain Reaction (PCR) and massively parallel,
next generation sequence provides a powerful and rapid
alternative. However, knowledge of gut-associated bacteria
in weevils derived from previous molecular studies has been
limited to a sparse and idiosyncratic collection of taxonomic
groups. Our project aims to survey the diversity and
understand the evolutionary histories of gut-associated
bacterial symbionts by sampling diverse representatives of
major lineages of weevils.
Background
• Investigate the diversity of gut bacterial symbionts from
a comprehensive sample of weevils.
• Detect symbiotic bacteria and examine their distributions
across different groups of weevils.
• Explore the patterns of bacterial OTU accumulation in
relation to sequencing depth.
• Determine the constituents of bacterial gut microbiota
and the relative abundance of bacterial groups.
Objectives
Figure 6. Habitus images of
representative weevil species
Conclusion
This project reveals that weevil gut
microbiotas are diverse and complex.
We have detected symbiotic bacteria
in weevils from a much greater
taxonomic breadth (Fig. 1) than
previously known. Gut microbiotas
are structured according to the
taxonomic groups of weevils. We
show that bacterial OTUs
accumulation rates vary greatly across
samples (Fig.2). We have found that
weevil gut microbiotas differ in alpha-
diversity (Fig. 3) and taxonomic
constituents (Fig. 5), but also exhibit
convergence to major groups of
bacteria (Figs 4 & 5). Future research
should investigate the diversity and
evolution of weevil-specific
endosymbiotic bacteria.
Subfamily
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Anthribinae
Apioninae
Baridinae
Ceutorhynchinae
Conoderinae
Cossoninae
Cryptorhynchinae
Curculioninae
EnDminae
Erirhininae
Hylobiinae
Hyperinae
Lixinae
MolyDnae
Orthognathinae
Rhyncophorinae
Trachelizinae
unplaced
Erwinia
(Enterobacteriaceae)
Sodalis
(Enterobacteriaceae)
Entomoplasmatales
(Mollicutes)
Comamonadaceae
(Betaproteobacteria)
Chloroplast
Enterococcus
(Enterococcaceae)
Wolbachia
(RickeSsiaceae)
RickeSsia
(RickeSsiaceae)
Enterobacteriaceae
Results
Figure 1. Frequency of the most abundant groups of bacteria in 18 weevil subfamilies, illustrating
that each subfamily has a distinct profile of bacterial abundance, and endosymbiotic bacteria
are widely present (e.g., Sodalis, Wolbachia, Rickettsia, Spiroplasma, and Curculioniphilus).
Figure 4. Relative abundance of bacteria at the phylum-
level in all samples, showing that Proteobacteria are
the most abundant phylum.
Figure 2. Rarefaction curves comparing the
number of OTUs with the number of sequence
reads in all samples. The samples show a
range of OTU accumulation patterns.
Figure 5. Relative abundance of bacteria at the
genus/family-level in all samples, showing that
the samples vary in constituents and relative
abundance of bacteria.
Figure 3. Alpha-diversity of bacteria OTUs in
weevil subfamilies. Most subfamilies host
~500-1500 OTUs, and Hylobiinae hosts the
highest numbers (~1200-3000) of OTUs.
Subfamily
Observed
OTUs
Acknowledgements
Funding was provided by a SOLS RTI postdoctoral
collaborative award and a NSF grant
(DEB-1155984). We thank Joseph Hunter for
assistance with specimen dissection, and Franz Lab
members for specimen donation.
Email: zgeng1@asu.edu, Twitter: @makoto_zhen