Colony introduction assays to study nestmate discrimination of the ant Pristomyrmex punctatus

Social animals utilise various communication methods to organise their societies. In social insects, nestmate discrimination plays a crucial role in regulating colony membership. Counter to this system, socially parasitic species employ diverse behavioural and chemical strategies to bypass their host’s detection. In this study, we tested whether such parasitic adaptations could be detected in the incipient stage of social parasitism that is observed as intraspecific phenomena in some social insects. The Japanese parthenogenetic ant Pristomyrmex punctatus harbours a genetically distinct cheater lineage which infiltrates and exploits host colonies. We found that intrusion of this intraspecific social parasite was defended by nestmate discrimination of host colonies without any behavioural strategies specialised in social parasitism. Most of the cheaters were eliminated through aggression by host workers that are typically observed against non-nestmates, resulting in a low intrusion success rate for the cheaters (6.7%). Our result contrasts with the expectation from interspecific social parasitism but rather resembles the intraspecific counterpart reported in Cape honeybees (Apis mellifera capensis), illustrating the role of nestmate discrimination in defence against the intrusion of intraspecific social parasites.

Methods
Experiment:

Colonies of Pristomyrmex punctatus were collected in Kihoku, Mie Prefecture, Japan. The colonies were maintained in the laboratory. The “colony introduction” assay (Roulston et al. 2003) was performed where a intruder individual (marked on the abdomen) was introduced into an experimental host colony. Intruders were (1) cheaters, (2) foragers, (3) nurses, and (4) nestmate nurses as negative conrtol. After introduction the experimental arena was video-recorded for at least 300 seconds. One hour and 24 hours after introduction, we checked inside the arena to observe the survival and location (inside or outside of the nest) of the intruder.

Analysis:

We analysed the video-recorded behaviours of the intruder and the host individuals during the colony introductions. Each video was assigned a random ID to blind the intruder types as much as possible. We scored the behaviours (0 to 4 and immobilised) by eye until 25 encounters with host individuals were made by an intruder. Statistical testing was all implemented in R.