Diversity and evolution of bacterial symbionts in weevils by 16s sequencing
Bacterial symbionts play diverse and critical roles in insects, such as provisioning nutrients, promoting temperature tolerance, conferring parasite resistance, manipulating reproduction, overcoming host defense, and most recently discovered, even in helping producing aggregation pheromones. Advances in high-throughput sequencing technologies are increasing the capacity to study endosymbiosis by orders of magnitude. We have thus entered a new age primed to unravel the fascinating phenomena of endosymbiosis in the most diverse eukaryotic organisms, the insects.
My project is addressing this challenge with a focus on beetles of the weevil superfamily Curculionoidea (aka weevils). Existing knowledge of weevil endosymbionts is scattered, but offers promising insights. At least three unique lineages of possibly obligate endosymbionts ("Candidatus Nardonella", "Candidatus Curculioniphilus" and SOPE [S. oryzae principal endosymbiont]) have been found in various weevils. Facultative endosymbionts – e.g., Wolbachia, Rickettsia, Sodalis and Spiroplasma – have also been recorded.
Previous surveys of bacterial symbionts in weevils had one common limitation--they had very sparse taxonomic sampling. In this project I am sampling four families or 17 subfamilies of weevils, which would be the largest taxonomic sample gathered for exploring the diversity of symbionts in weevils.
Experiments have been completed and procedures are as follows: (1) weevil specimens were dissected and gut contents recovered; (2) samples were subjected to bead-beating (see advantages) and then DNA was isolated with the Qiagen DNeasy Blood & Tissue kit; (3) the 16s gene (V4-V5 region, positions 515-909) was amplified using PCR with barcoded primers (for sample-indexing); (4) PCR products were gel-purified using the Freeze 'N Squeeze spin columns (Bio-Rad); (5) PCR products were quantified on a Nanodrop and normalized using the SequalPrep™ Normalization Plate Kit (ThermoFisher Scientific); (6) normalized PCR products were pooled into a single sample, which was processed for library preparation; and finally (7) sequenced on an Illumina Miseq (paired end) platform.
We have performed data exploration and preliminary analyses based on the 11 million + assembled sequence reads. Some potentially significant insights we gained from the analyses are (1) "Ca. Nardonella", "Ca. Curculioniphilus" and SOPE, previously found only from weevils, were found in all weevil specimens examined in the current study, indicating their conservation in weevils; (2) these bacteria were also detected in other related weevils (Chrysomeloidea, Tenebrinoidea and Cleroidea), and hence may be more widespread than previously thought; (3) a single weevil generated several 16s sequences that belonged to the same bacterial lineage (e.g., "Ca. Nardonella") but were found to be polyphyletic, indicating horizontal transfer or duplication and selective extinction; and (4) other 'generic' symbionts such as Wolbachia, Rickettsia, and Spiroplsma were also found across nearly all samples.
Current analyses are directed at testing co-evolutionary scenarios, examining for possible horizontal transfer or duplication of symbionts, and comparing diversity across samples.
I am collaborating with Dr. Patrick Browne, former postdoc in Dr. Hinsby Cadillo-Quiroz's lab, Ms. Zhen Geng and Andrew Johnston on this project. Ms. Geng is an undergraduate student researcher and worked closely with me. She has done the heavy lifting of performing most of the experiments. Dr. Browne leads the bioinformatics part of the project and Mr. Johnston recently joined the project to help with various aspects of the analyses.