DOI: 10.5061/dryad.59zw3r2h2
title: Colony introduction assays to study nestmate discrimination of the ant Pristomyrmex punctatus

description: 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.
description: <em>Experiment:</em> Colonies
 of <em>Pristomyrmex punctatus</em> 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. <em>Analysis:</em>
 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. 
description: # Colony introduction assays to study nestmate discrimination of the ant
 *Pristomyrmex punctatus*
 [https://doi.org/10.5061/dryad.59zw3r2h2](https://doi.org/10.5061/dryad.59zw3r2h2) Behavioral scores and associated measures observed in encounters between intruders (cheaters, foragers, nurses, and nestmates) and host individuals of *P. punctatus*. ## Description of the data and file structure File structure: ``` Data1.csv ## Results of behavioral scoring, line per experimental colony Data2.csv ## Results of behavioral scoring, line per observation (25 obs. per colony; clarifying behavioral correspondences) Code.R ## An R code to generate the statistical results Movies.zip ## Original movie files that recorded the behavioral assays |- ex\d{3}.mp4 (each stored in mp4 format) ``` Column description of the Data1.csv is provided below: ``` Date: YYYYMMDD of the onset of experiment. ID: Randomly assigned number for blind tests. Intruder: ID of intruder-source colony (given in Table S2). Home: Source colony of host workers. type: Type of the introduced intruder: cheater, forager, nurse, and nestmate. Comb: Combination of intruder-source and host-source colonies. first: Binary to select the first trial of experiments for each intruder-source and host-source colony combination and for each type of intruder, except for the negative control (three trials per experiment); 1 = First trial or negative control, 0 = Others final: Binary to select the last trial of experiments for each intruder-source and host-source colony combination and for each type of intruder, except for the negative control (three trials per experiment); 1 = Last trial or negative control, 0 = Others surv1: Binary to indicate survival of the intruder 1 hour after the introduction; 1 = Alive, 0 = Dead, NA = undetermined due to the accidental removal of the paint mark or unintended accidents. loc1: Binary to indicate location of the intruder 1 hour after the introduction; 1 = Inside the nest, 0 = Outside the nest, NA = undetermined due to the accidental removal of the paint mark or unintended accidents. mark1: Binary to indicate presence of the paint mark on the intruder 1 hour after the introduction. 1 = Present, 0 = Absent, NA = undetermined due to unintended accidents. surv24: Binary to indicate survival of the intruder 24 hour after the introduction; 1 = Alive, 0 = Dead, NA = undetermined due to the accidental removal of the paint mark or unintended accidents. loc24: Binary to indicate location of the intruder 24 hour after the introduction; 1 = Inside the nest, 0 = Outside the nest, NA = undetermined due to the accidental removal of the paint mark or unintended accidents. mark24: Binary to indicate presence of the paint mark on the intruder 24 hour after the introduction. 1 = Present, 0 = Absent, NA = undetermined due to unintended accidents. h0: Frequency with which the score 0 = ignore was observed in host workers, out of 25 observations. h1: Frequency with which the score 1 = touch was observed in host workers, out of 25 observations. h2: Frequency with which the score 2 = avoid was observed in host workers, out of 25 observations. h3: Frequency with which the score 3 = aggression was observed in host workers, out of 25 observations. h4: Frequency with which the score 4 = fighting was observed in host workers, out of 25 observations. hi: Frequency with which the score immobilised was observed in host workers, out of 25 observations. i0: Frequency with which the score 0 = ignore was observed in the intruder, out of 25 observations. i1: Frequency with which the score 1 = touch was observed in the intruder, out of 25 observations. i2: Frequency with which the score 2 = avoid was observed in the intruder, out of 25 observations. i3: Frequency with which the score 3 = aggression was observed in the intruder, out of 25 observations. i4: Frequency with which the score 4 = fighting was observed in the intruder, out of 25 observations. ii: Frequency with which the score immobilised was observed in the intruder, out of 25 observations. insi: Duration in seconds for which the intruder was located inside the nest, during 300 seconds from introduction. time25: Duration in seconds taken for the intruder to finish her 25th behaviour ``` Column description of the Data2.csv is provided below: ``` ID: Randomly assigned number for blind tests. Intruder: ID of intruder-source colony (given in Table S2). Home: Source colony of host worker. type: Type of the introduced intruder: cheater, forager, nurse, and nestmate. encounter: Number of behavioural interactions between the host workers and the intruder hbeh: Behavioural score of host workers; 0 = ignore, 1 = touch, 2 = avoid, 3 = aggression, 4 = fighting, immobilised = being immobilised. ibeh: Behavioural score of the intruder; 0 = ignore, 1 = touch, 2 = avoid, 3 = aggression, 4 = fighting, immobilised = being immobilised. ```