Background

Eusocial insects (ants, bees, wasps, and termites) live in colonies in which all individuals have a high genetic relatedness, while only one or few individuals reproduce (Chapman et al., 2000; Trontti et al., 2005; Loope, 2015). The reproductive (queens and also kings in termites) and non-reproductive (workers) castes in a colony display different phenotypic traits including differential behavior, reproduction, metabolism, and lifespan, a phenomenon known as polyphenism (Simpson et al., 2011; Kapheim et al., 2012). Eusocial insects offer a valuable experimental model to compare gene expression between castes and to identify the key genes as causal factors determining differential phenotypes at the organismal level (Colgan et al., 2011; Bonasio et al., 2012).

Harpegnathos saltator (commonly known as the Indian jumping ant) is an ant species originating from India. These ants display an unusual feature: individuals can transition from non-reproductive workers to reproductive pseudo-queens (also called gamergates) (Liebig et al., 2000; Monnin and Peeters, 2008; Sieber et al., 2021a). When the queen is absent from a colony, workers can switch their caste and show queen-like traits: minimal movement, staying inside the nest, active ovaries, and production of eggs. In contrast, some workers show active movement in the nest to take care of the progeny, while others hunt and forage. Furthermore, workers have small ovaries and rarely produce eggs.

A colony with gamergates is as stable as a queen colony (Opachaloemphan et al., 2021; Penick et al., 2021). Additionally, gamergates can be experimentally converted back to workers (to distinguish, they will be referred to as revertants) (Penick et al., 2021). This process is associated with the reversion of phenotypic traits: revertants display worker-like physiology, behavior, and aging (Penick et al., 2021; Yan et al., 2022). With a high-quality genome (Bonasio et al., 2010; Shields et al., 2018), multiple studies have been performed in H. saltator to analyze caste-specific transcriptomes and methylomes at tissue and/or single-cell levels (Bonasio et al., 2012; Sheng et al., 2020; Sieriebriennikov et al., 2021). H. saltator is one of the three species (along with Oocerea biroi and Solenopsis invicta) in which reverse genetics was applied in ants using the CRISPR-Cas9 system (Trible et al., 2017; Yan et al., 2017; Chiu et al., 2020). This experimental paradigm has helped to identify cellular and molecular processes required for the establishment of caste identity (Gospocic et al., 2021; Opachaloemphan et al., 2021) and determine the role of neuropeptides, hormones, and transcription factors in regulating caste-specific behaviors, and the role of insulin signaling in reproduction and longevity (Penick et al., 2014; Gospocic et al., 2017 and 2021; Yan et al., 2022). Here, we provide a detailed protocol explaining the experimental framework we have used on H. saltator to induce caste transition and reversion, the identifiable and quantifiable parameters to define each caste, and the techniques for tissue dissection and staining.