Three decades of analyzing Wolbachia bacterial endosymbionts in arthropods: Trends and gaps.

Wolbachia are the most pervasive bacterial endosymbionts yet described, infecting half of all arthropod species. These bacteria trigger outstanding phenotypes in their hosts including cytoplasmic incompatibility (CI), which has also been used as a mechanism to control pest species. Here, we analyzed peer-reviewed articles published in the 30 years from 1995 to 2024. Our results show that most studies continue to use traditional methods such as PCR and Sanger sequencing, approaches that remain sufficient and appropriate in many cases, particularly when working with well-characterized strains or applied systems. Only a smaller proportion of studies have employed newer genome-based techniques, which are increasingly important for exploring Wolbachia diversity and uncovering novel mechanisms. Research has focused mainly on a small number of insect groups and a limited set of Wolbachia strains. In addition, most work focuses on CI, especially since the discovery of cif genes related to this trait. Although the use of Wolbachia in pest and disease control is expanding, other possible effects and interactions with different microbes remain less explored. Our analysis shows how the field has advanced through some key discoveries, while many studies continue to use established approaches and concentrate on a limited set of hosts and strains. To make further progress, we suggest using a wider range of methods and sampling a broader set of hosts, alongside integrating genetic tools with studies of natural populations. This work outlines clear areas where more research is needed and points to ways the field can develop a fuller understanding of Wolbachia and its roles in nature.