DOI: 10.5061/dryad.h18931zj3
title: Data from: Age-based changes in kairomone response mediate task partitioning in stingless bee soldiers (Tetragonisca angustula)

description: Collective defense is one of the most ubiquitous behaviors performed by
 social groups. Because of its importance, complex societies may engage a
 set of defensive specialists, with physical and/or neurological attributes
 tuned for defense against specific invaders. These strategies must be
 balanced, however, with the need to flexibly respond to different threat
 levels and sources. Insect societies rely heavily on olfaction for
 detecting and communicating in the context of defense. We therefore asked
 whether threat detection via olfaction is specialized towards
 invader-specific cues and how this may be integrated into defense task
 specialization. Colonies of the stingless bee, Tetragonisca
 angustula, deploy a morphologically distinct sub-caste of larger-bodied
 workers (soldiers) for colony defense. These soldiers transition between
 two different guarding tasks as they age, progressing from guarding in a
 hovering position near the nest entrance to guarding in a standing
 position on the nest entrance tube. Hovering and standing guards intercept
 different types of invaders: primarily heterospecific versus conspecific,
 respectively. We asked whether hovering and standing guarding behaviors
 were modulated by differential sensitivity to invader-associated olfactory
 stimuli; then we compared their responses to these cues to those of
 smaller workers that perform predominantly non-defense tasks. We exposed
 bees under both field and lab conditions to citral, a kairomone produced
 by an obligate heterospecific nest robber, primarily intercepted by
 hovering guards. Consistent with their roles, hovering guards were more
 likely to move towards citral than were either standing guards or
 small-bodied bees within a Y-maze. We also presented guards at field nests
 with dummies of conspecific versus heterospecific invader types, varying
 whether they included citral odors. Standing guards were more responsive
 to conspecific intruder scenarios than hovering guards, but heterospecific
 response differed by presence of citral. Standing and hovering guards
 responded in similar proportions when citral was absent, but the addition
 of citral produced a marginally non-significant reduction in standing
 guard participation. Our results potentially demonstrate differentiated
 cue-specific responses that correspond to morphological task
 specialization and age polyethism in these eusocial societies.
description: All field and lab manipulations were conducted using subjects
 from naturally occurring nests in Gamboa, Panama (9.12° N, 79.70° W). Data
 were gathered in January 2018 and February 2019. Task group assignments
 for each experiment were made using established behavioral criteria
 (Baudier et al. 2019). Bees hovering at the entrance for longer than 20 s
 while facing inwards towards the flyway were considered hovering guards.
 Bees standing on the wax and resin nest entrance tube for longer than 20 s
 while facing towards the entrance opening were considered standing guards.
 Minors (smaller-bodied worker bees that do not typically defend the
 colony) were collected while in the task of foraging because this task is
 performed by small workers during 3–4 weeks since adult emergence, making
 as close as possible an age-matched comparison group to soldiers that
 perform the task of hovering in approximately the third week following
 adult emergence and the task of standing guarding in approximately the
 fourth week following adult emergence (Hammel et al. 2016; Baudier et
 al. 2019). A bee was considered a forager if it exited the nest and flew
 immediately away from the nest entrance (unlike hovering guards) but
 carried no detritus material (unlike dump workers). In a previous study,
 subject weights confirmed that hovering and standing guards gathered using
 this method were significantly larger in body size than collected foragers
 (Baudier et al. 2019). Y-maze olfactory response
 bioassays We conducted two types of Y-maze assays. The
 first assessed affinity of bees to citral, and the second assessed
 starvation-motivated bee affinity to a nutrition source (mixed pollen and
 honey). This allowed for the comparison of defense-specific olfactory
 response as well as appetitive olfactory acuity across the 3 focal task
 groups: minor bees, standing guards, and hovering guards. All Y-maze
 assays were performed in an ambient lab with relative humidity between 81
 and 100% and temperature ranging from 24 to 30 °C. To
 test the affinity of bees in different task groups for citral, we
 introduced bees to Y-mazes with citral at the end of one arm. A total of
 54 naïve individual bees were used (6 bees × 3 task groups × 3 colonies
 per assay). Subject bees were first collected from nest entrances by
 aspirator into 38 × 84 mm vented plastic tubes and placed into a dark
 environment for 15 min prior to each citral Y-maze assay in order to allow
 bees to calm from any alarm caused during collection. We used 3D-printed
 Y-mazes constructed from white PLA plastic (MakerBot Industries, LLC) and,
 with an interlocking, transparent acrylic lid to enable observations.
 Y-maze arms were 10 cm long, 1 cm wide, and 1 cm tall. A 2.5 × 1 × 1 cm
 entrance vestibule adjoined to arms, each offset at 45° (90° angle between
 the arms). A quantity of 2 μl of 95% citral (Sigma-Aldrich, CAS Number
 5392-40-5) was placed on a 4 × 4 mm filter paper, while the other arm was
 left with an unscented filter paper. This small dose was based on previous
 studies which approximated citral quantity in natural contexts, given that
 1 ml of a 1% citral solution was sufficient to illicit defensive
 behavioral responses in this species (Jernigan et al. 2018). Air flow was
 provided in the maze using a pump to inhibit odor diffusion in the
 non-treatment arm. We set the air flow to 166 ml/s, strong enough to
 prevent the scent of citral from permeating the opposite arm (according to
 human olfaction), but mild enough to not inhibit the smallest bees from
 navigating through the maze. To eliminate directional bias, citral was
 alternated equally between the left and right arms of the Y-maze. Each
 replicate of the assay started when the subject bee was placed in the
 vestibule and ended only when the scented filter paper was contacted by
 the bee (all bees in the study touched the scented filter paper in under 5
 min of observation). The time until each bee contacted the scented filter
 paper and each bee’s first arm choice (towards versus away from odor) were
 recorded. A subject’s first walk &gt; 5 cm down an arm from the
 vestibule was considered its first arm choice. Y-mazes were scrubbed with
 soap and water, rinsed with water, then rinsed with ethanol, and allowed
 to air dry between replicates. Similar Y-maze materials and cleaning
 protocols have been used in previous olfactory studies with hymenopteran
 subjects, without apparent complications associated with scent
 contamination (Carcaud et al. 2009; Provecho and Josens 2009; Arenas and
 Farina 2012; Duell 2018). To test for differences in
 general olfactory acuity, we similarly compared the 3 groups
 (minors/foragers, hovering guards, and standing guards; N = 54 naïve bees;
 6 bees × 3 task groups × 3 colonies per assay) in their ability to locate
 the smell of food reward in the same Y-maze setup when starved/water
 deprived. The same collection and Y-maze procedures were used for this,
 except that bees were deprived of food or water in darkened, vented tubes
 for 55 min prior to Y-maze introduction (to motivate food search), and a
 liquid food reward mixture (40% honey and 20% pollen of locally
 farmed <em>Apis mellifera</em>, 40% water) was applied to one
 end of a Y-maze arm as the focal odor source. This mixture was wiped
 lightly along the last 0.5 cm of one Y-maze arm. We recorded time until
 the liquid food mixture was contacted and the bee’s first arm
 choice. All analyses were conducted in R version 3.4.4
 (R Core Team 2020). Variation between task groups in time until solving
 either food or citral odors in the Y-maze was compared using mixed model
 survival analyses due to the non-parametric distribution of the data. In
 each case, we used a Cox proportional hazard analysis that included and
 colony ID as a random factor and task group (forager/minor, hovering
 guard, standing guard) as a fixed predictor of time until bees found the
 odor source within the mazes using the coxme command in the package
 “coxme” (Therneau 2015). We then followed with a post hoc Tukey HSD test
 to compare among the three task groups. To test for
 task group differences in first Y-maze arm choice, we fit a binomial mixed
 effects model with arm choice as the response variable, task group
 (hoverer, stander, forager) as a fixed factor, and colony as a random
 factor using the glmer function (Bates et al. 2015) and then performed a
 Type-II analysis on the fitted model to test significance of fixed
 effects. This was done separately for the citral and starved food choice
 assays. At-nest manipulations To
 test whether lab-observed patterns in citral reaction were observable in a
 more naturalistic field setting, we measured guard responses to
 citral-coated dummies at nest entrances. At each subject colony nest
 entrance, we introduced two treatment mock invaders and two controls. The
 first treatment was a freshly freeze-killed worker of the stingless
 bee Partamona peckolti, an environmentally abundant competitor in the area
 that is black in body color (like <em>Lestrimelitta</em>) but
 does not use citral to recruit raids. The second treatment was an obligate
 nest-robber kairomone dummy consisting of a black polymer clay figure of
 roughly equal size, shape, and color (black) as the
 average <em>Lestrimelitta danuncia</em> worker and dipped in
 95% citral (approximately 0.02 ml of citral). <em>Tetragonisca
 angustula</em> respond similarly to actual invaders as they do to
 such color- and odor-matched dummies (Bowden et al. 1994).
 Using this design, if the division of labor between hovering and
 standing guards is (at least in part) driven by differences in
 responsiveness to olfactory cues, then we predicted that a higher number
 of hovering guards would respond to the high-citral dummy, compared with
 the responsiveness of standing guards. For reference, we also included a
 positive control for eliciting standing guard attack behavior: a
 freeze-killed non-nestmate T. angustula worker. Each intruder dummy was
 harnessed with wire onto the end of a dowel before being presented at nest
 entrances. An empty wire was also used as a negative control for attack
 behavior alongside each of the three treatment dummy invaders. A negative
 result from the empty wire control would confirm that there was no
 olfactory contamination of instruments and that the effect of
 non-olfactory disturbance at the nest (human presence, vibrational
 disturbance, etc.) on the aggressive behaviors of interest was
 minimal. Each mock intruder or control was brought
 directly towards the nest entrance tube (0° angle) and contacted the edge
 of the tube for 2 s. During each trial, we recorded number of attackers
 (predominantly soldiers) and each attacker’s guard type (hoverer versus
 stander). This is a standard method for eliciting and measuring defensive
 guard attack in this species (Bowden et al. 1994). Attacks were defined as
 contact with a mock invader for greater than 2 s. Each mock intruder and
 wire were discarded and replaced after use to prevent cross contamination
 of scents. Treatments and control wire trials were conducted 15 min apart
 at each subject colony and were presented in a pseudo-random order. A
 total of five colonies were tested in this manner, and each colony was
 repeatedly tested on 3 different days. We used a mixed
 effect model approach to repeated-measures analysis comparing the effects
 of guard type and invader type on total responses. Number of bee responses
 was first z-transformed to improve normalcy of distributions. Next, we fit
 a linear mixed effect model (lme4 package, lmer function) (Bates et
 al. 2007; Bates et al. 2015), which included guard type (hovering vs.
 standing), mock intruder treatment, and the interaction between the two as
 fixed predictors of number of bee responses (z-transformed number of bees
 contacting model for at least 2 s) and included colony as a random factor.
 We used a Type-II Wald chi-square test of this model to test significance
 of fixed factors. We then followed this analysis with post hoc pairwise
 comparisons of standing versus hovering guard responses within each
 intruder treatment (colony kept as a random factor), using multiple
 Type-II Wald chi-square tests with adjusted p values according to these
 multiple comparisons using the Benjamini and Hochberg method (Benjamini
 and Hochberg 1995).<br> <br> References
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description: <strong>At nest manipulations.csv</strong>
 This file contains all at-nest manipulation data associated used
 in this study.  Headder definitions are as follows.
 ColonyID - unique identifier for each of 5 naturally occuring
 colony/nest entrances used in this part of the study.
 Rep - replicate ID (repeated across 3 days, 3 levels: day 1, 2,
 or 3) guardTYPE - type of responding soldier (2 levels:
 hovering guard, standing guard) guardRESP - number of
 each guard type identified in "guardTYPE" responding to each
 invader type   <strong>food
 detection Y-maze.csv </strong> & <b>alarm detection
 Y-maze.csv</b> These files contain all y-maze
 data analyzed in this study (other than first arm choice, which is
 presented in tables within the manuscript). They contain data for speed at
 which different task group bees detected food and citral
 (respectively) within Y-mazes. Headder definitions are as
 follows: BeeID - individually unique identifier of each
 naive bee used in this study.  ColonyID - unique
 identifier for each of 3 naturally occuring colony/nest entrances used in
 this part of the study. Task - task group of each bee
 (3 levels sampled in each colony: hovering guard, standing guard,
 & foragers) cens - censor data used in the
 performed survival analysis. Because all bees found the odor sources in
 the maze eventually, these values are all "1"