Submission Type
Oral Presentation
Abstract
Insects are hosts to diverse communities of other organisms. These communities can include microbes such as bacteria, yeast, and protists, as well as multicellular organisms such as nematodes and mites. The prokaryotic associates of many insects have been well-studied and are often investigated by amplifying and sequencing the gene for the 16S ribosomal subunit using universal primers.
However, the eukaryotic associates of many insects remain poorly characterized, and this is in part due to the difficulty in utilizing 18S (the eukaryotic analog for 16S) for eukaryote detection. Because the insect host is also a eukaryote and makes up the bulk of biomass in each sample, attempts to amplify 18S using universal primers results in exclusive or near exclusive amplification of the host insect sequence and hindered detection of associate sequences that were at low initial abundance.
Here I present the development and testing of “blocker primers” as a method to preferentially suppress PCR amplification of host insects while allowing amplification of eukaryotic associates by universal primers. I show that blockers specific to host insect order are successful at reducing the proportion of host reads from >90% of sequenced reads to <2%, resulting in an increased proportion of sequenced reads from eukaryotic associates. This paves the way for the first ever broad screens of eukaryote symbiont diversity across a taxonomically diverse range of insects.
Included in
Biodiversity Commons, Biology Commons, Ecology and Evolutionary Biology Commons, Entomology Commons
Development of host-selective, PCR-inhibiting oligos as a tool to investigate the eukaryotic symbionts of insect hosts
Insects are hosts to diverse communities of other organisms. These communities can include microbes such as bacteria, yeast, and protists, as well as multicellular organisms such as nematodes and mites. The prokaryotic associates of many insects have been well-studied and are often investigated by amplifying and sequencing the gene for the 16S ribosomal subunit using universal primers.
However, the eukaryotic associates of many insects remain poorly characterized, and this is in part due to the difficulty in utilizing 18S (the eukaryotic analog for 16S) for eukaryote detection. Because the insect host is also a eukaryote and makes up the bulk of biomass in each sample, attempts to amplify 18S using universal primers results in exclusive or near exclusive amplification of the host insect sequence and hindered detection of associate sequences that were at low initial abundance.
Here I present the development and testing of “blocker primers” as a method to preferentially suppress PCR amplification of host insects while allowing amplification of eukaryotic associates by universal primers. I show that blockers specific to host insect order are successful at reducing the proportion of host reads from >90% of sequenced reads to <2%, resulting in an increased proportion of sequenced reads from eukaryotic associates. This paves the way for the first ever broad screens of eukaryote symbiont diversity across a taxonomically diverse range of insects.
Comments
Dr. Andrew A. Forbes, University of Iowa