High-Throughput Amplicon Sequencing for Analyzing Microbial Communities of Insects

Insects represent more than 80% of all described species on the planet. This diversity is a result of millions of years of evolution, during which insects have colonized nearly every habitat. Their success is partly due to their ability to form symbiotic relationships with a wide variety of other organisms, especially microorganisms. Identifying and characterizing associated microorganisms are crucial to understanding the complexity and dynamics of these symbiotic relationships. To date, advancements in sequencing technologies that provide large sequence data sets have become ideal tools for characterizing insect microbiomes, including information about non-cultivable microorganisms commonly found in insects. Despite the growing number of studies focused on insect microbiome characterization, there are few protocols detailing methodological procedures for fieldwork, DNA extraction, and data processing. Here, we present an overview of the characterization of insect-associated bacterial communities. We cover best practices for data interpretation and visualization, including alpha and beta diversity analyses, community composition profiling, and statistical testing to identify microbial associations of insects.