BugBook: How to explore and exploit the insect-associated microbiome

Abstract Large scale insect farming is exploring routes to enhance the efficiency, stability, and safety of the bioconversion of low-value substrates into insect-derived building blocks for food, feed, and fertiliser. Along with optimising insect rearing conditions and genetics, the insect microbiome is fundamental for the physiology, development, and adaptation of its host to various environmental conditions. To efficiently explore and exploit this ecosystem, a thorough understanding of its composition, function, and dynamics is required. This article aspires to provide a synopsis of the methodologies used to probe the insect-associated microbiome, primarily focusing on industrially relevant insect species. Key considerations for sample timing, selection, storage, and processing are discussed, emphasising the importance of standardised approaches to facilitate cross-study comparisons and enhance reproducibility. Marker gene and shotgun metagenomic sequencing are contrasted as means to investigate microbiome features, touching upon their respective (dis)advantages and potential use cases. Cultivation-based methods are essential for functional characterisation and translating the potential of insect-derived microorganisms for industrial applications. Direct isolation and enrichment cultures, along with anaerobic and aerobic cultivation techniques, are discussed as well. Methods to engineer microbiomes, such as axenic rearing and synthetic community assembly, have developed as powerful tools for exploring the role of specific microbes in host physiology. Beyond these approaches, metabolomics and metaproteomics are emerging as insightful techniques to dig deeper into microbiome functionality and host-microbe interactions. This article provides a multifaceted outline for researchers investigating the insect-associated microbiome and emphasises the importance of standardised methodologies and reporting for advancing the field.