How to study enhancers in non-traditional insect models.

Transcriptional enhancers are central to the function and evolution of genes and gene regulation. At the organismal level, enhancers play a crucial role in coordinating tissue- and context-dependent gene expression. At the population level, changes in enhancers are thought to be a major driving force that facilitates evolution of diverse traits. An amazing array of diverse traits seen in insect morphology, physiology and behavior has been the subject of research for centuries. Although enhancer studies in insects outside of have been limited, recent advances in functional genomic approaches have begun to make such studies possible in an increasing selection of insect species. Here, instead of comprehensively reviewing currently available technologies for enhancer studies in established model organisms such as , we focus on a subset of computational and experimental approaches that are likely applicable to non- insects, and discuss the pros and cons of each approach. We discuss the importance of validating enhancer function and evaluate several possible validation methods, such as reporter assays and genome editing. Key points and potential pitfalls when establishing a reporter assay system in non-traditional insect models are also discussed. We close with a discussion of how to advance enhancer studies in insects, both by improving computational approaches and by expanding the genetic toolbox in various insects. Through these discussions, this Review provides a conceptual framework for studying the function and evolution of enhancers in non-traditional insect models.