UC Riverside

In the last two decades, the field of molecular entomology has seen a shift toward next-generation sequencing techniques as a means of uncovering genetic and developmental processes. However, the standardization of methods is not well-established, and studies for insect-fungus consortia lack established protocols for advanced molecular techniques and downstream analysis compared to approaches applied in model systems involving insect-bacteria interactions. To investigate insect-microbe interactions, RNA sequencing and analysis is often used to identify genes involved in the symbiosis. But such protocols do not often consider insect-fungus systems, which vary significantly in community member abundance and/or fail to describe the details of the process from collection to data processing. This paper will introduce a comprehensive approach for RNA sequencing using two non-model insect-fungus consortia, which lack established, published protocols seen in model systems: the ambrosia beetle mutualism and cicada Massospora parasitism. The protocol includes a detailed TRIzol RNA extraction and quantification, RNA sequencing, and data processing using Nextflow pipeline software. Validation of a range of symbiotic interactions from mutualistic to parasitic is considered to justify this procedure to be utilized in a range of insect-fungus interactions with varied abundances and host interactions. Key features • Stepwise protocol for RNA extraction of samples containing insect and fungal tissue. • Novel dissection technique for beetle pupae. • Acquisition of transcriptomes of both host and symbiont with one protocol. • Direct comparisons of transcriptomes across life stages, stages of symbiosis, and/or by treatment.