Background

Cuticular hydrocarbon (CHC) is the generic term for dozens of hydrocarbons on the surface of insect cuticles. CHC protects insects from desiccation and functions as a semiochemical (Howard and Blomquist, 2005; Sturgis and Gordon, 2012). In particular, the role of CHC in the ant social system has been studied in detail. For example, CHC is used to discriminate between nestmates and non-nestmates (Howard and Blomquist, 2005; Sturgis and Gordon, 2012) and inhibit workers’ oviposition (Howard and Blomquist, 2005; Kocher and Grozinger, 2011). Furthermore, some parasitic species take advantage of this CHC-based nestmate discrimination mechanism to invade the nests of host ants (Dettner and Liepert, 1994; Lenoir et al., 2001; Howard and Blomquist, 2005; Akino, 2008; de la Mora et al., 2020). For example, when the newly mated Polyrhachis lamellidens and Polyergus samurai queens invade a host colony, they perform rubbing behavior against the host worker or kill the host queen. These behaviors help the newly mated queen to disguise her CHC profile and match that of the host ants. By disguising the CHC profile of the host ants, the newly mated queen is recognized as a nestmate, allowing her to achieve the early stages of social parasitism (Tsuneoka and Akino, 2012; Iwai et al., 2022).

Behavioral tests and bioassays using ants, as well as profiling and quantifying CHC, are potent approaches to understanding such nestmate discrimination and chemical disguise behavior. A highly efficient, stable, and reproducible extraction method for CHC is essential for this purpose. In addition, reproducing the chemical disguise behavior of socially parasitic ants in the laboratory is necessary. In this paper, we describe a method for CHC extraction and measurement, as well as the induction of chemical disguise by a socially parasitic ant under laboratory conditions, which has been established as an actual experimental system, using P. lamellidens and its host Camponotus japonicus as examples, referring to the practical techniques used in our previous study (on Polyrhachis and Camponotus species) (Iwai et al., 2022; Kurihara et al., 2022). In addition, we describe the method for the tracing assay using artificial isotopic substances to confirm the transition of the CHC profile from the host species to a socially parasitic ant.

First, the artificial isotopic substance is applied to the host worker; then, the socially parasitic ant performs the chemical disguise against the host worker. The crude extract, containing mainly CHC and polar compounds, is obtained from an anesthetized ant with hexane. Then, CHC is separated to remove other substances using silica gel. The fractionated product is concentrated by volatilizing the solvent with N₂ gas. A portion of this concentrate is then used for measurements in the gas chromatographymass spectrometry (GC-MS) system. As a result, several ant-derived CHC peaks are detected. This CHC extraction protocol is mainly for large species such as Polyrhachis and Camponotus, although it should be possible to use for small species by adjusting the number of ants used per sample. In addition, since the main targets of CHC profile disguising in socially parasitic ants are adult host workers and host queens, the use of artificial isotopic substances described in this paper should also be applicable to other species.