DOI: 10.5061/dryad.vx0k6dk5c
title: Data from: Social immunity in a supercolonial invasive ant: Nest structure confers immune function

description: In animals, group living comes at the cost of increased pathogen exposure.
 In kin groups, social immune behaviors offset that cost and reach their
 most complex expression in eusocial insect societies. In the nests of
 these societies, collective social behaviors can modify patterns of
 individual interactions across space, reducing the ability of pathogens to
 reach the reproductive core of the colony (organizational immunity). To be
 effective, these behaviors must separate infected and uninfected
 individuals, implying that the efficacy of social immune behaviors may
 depend upon nest structure. The role of nest space has received little
 attention, and most knowledge of social immune behavior in social insects
 is based on the study of generalist entomopathogenic fungi. We examine the
 social immune behaviors involved in the interaction between the
 supercolonial, invasive tawny crazy ant (Nylanderia fulva), and its
 specialist, intracellular, microsporidian pathogen Myrmecomorba
 nylanderiae, to ask how nest structure influences social immunity. By
 manipulating nest structure, we demonstrate that preventing pathogen
 transmission to the colony core requires a multi-chambered nest. Without
 which, social immune function was lost, and disease transmission was
 universal. To understand how nest space enhances social immune efficacy,
 we first confirm that workers within tawny crazy ant nests form spatially
 and behaviorally segregated social sub-networks. We then find that
 infected ants introduced into the colony core migrate to the colony
 periphery, while uninfected ants do not. Behavioral tests indicate that,
 despite the infection being internal, uninfected ants can detect the
 infection status of a worker; thus, behaviors enforcing spatial
 segregation could be triggered by either party. Additionally, infected
 ants alter the behavioral tasks they perform, assuming more corpse removal
 tasks, particularly infected corpse removal, and reducing their efforts in
 foraging and brood care. With some exceptions, the social immune behaviors
 expressed by this supercolonial ant in response to microsporidian
 infection correspond to immune defense behaviors employed to defend
 against generalist entomopathogenic fungi. These behaviors appear to be
 conserved, generalized responses to pathogen infection among social
 insects.
description: <em><strong>Methods</strong></em> We test whether nest structure alters immune function. Then, we examine the mechanisms that contribute to this effect. First, we evaluate whether workers from uninfected colony fragments occupy spatially distinct social networks shortly after moving into a new nest. Second, we analyze how these spatially defined subnetworks differ in interaction types and task profiles. Third, we test whether infection status changes how workers segregate amongst these subnetworks. Fourth, we assess whether infection influences the interactions workers engage in or the tasks they perform, and finally, we evaluate whether infection status affects how aggressively workers interact. <strong>Infection Assessment: Colony Fragments</strong> The infection status of colony fragments was determined by homogenizing groups of 20 workers in 300 µl of DI H2O, fixing and staining a 0.3 µl aliquot of the tissue homogenate, and counting the Type II DK M. nylanderiae spores present [1].  It is not possible to artificially infect individual adult workers, as they acquire the infection as larvae, nor can we assess the infection status of individual workers non-destructively. Introduced ants were assigned the infection status of their natal colony fragments.  Spore counts of individuals from colony fragments with similar spore burdens show consistently high infection prevalences, with 85 to 95% of workers spore-positive with variable infection intensities. A fraction of the infected workers used in assays were likely uninfected or had low-level infections. Therefore, tests herein may underestimate differences between infected and uninfected workers. <strong>Multi-chamber nest-array</strong> We used a multi-chambered nest array to test how nest structure affects social immune function; to characterize both the social networks of colony fragments absent introduced individuals, as well as the behaviors and tasks assumed by infected and uninfected ants introduced into the arrays; and finally to evaluate how infected and uninfected ants assort spatially. Nest-arrays provide a multi-chamber structure allowing the ants to sort themselves spatially while keeping the queen and brood in the chamber farthest from the outside environment. <strong>Influence of nest structure on infection transmission</strong> To see if nest structure affects whether a colony fragment can resist infection, we introduced infected ants into nest-arrays and simple nest boxes. Simple boxes (24 x 14 x 9 cm) were translucent plastic with a water tube for the queen and brood. Nest-arrays had 396 cm<sup>2</sup> of floor area, and nest boxes had 336 cm<sup>2</sup>. In both, the ants clustered in a small part of the nest area, making inter-individual spacing insensitive to slight differences in nest area. Uninfected colony fragments, with 600 workers, two queens, and 1/8 cm³ of brood (including eggs and larvae, but excluding pupae), were housed in nest-arrays or simple nest boxes. Ants housed within each were termed ‘resident,’ while introduced ants were ‘aliens.’  Aliens originated from local-supercolonies far from the collection site of resident fragments. Three treatments were applied: 1) infected-alien workers introduced into the outer chamber of the nest array; 2) infected-aliens introduced into the queen chamber; and 3) infected-aliens introduced into a simple nest box. Pupae were collected weekly for infection testing. All available pupae were harvested and tested to confirm infections arose from direct transmission by introduced ants. <strong>Social networks of resident ants in nest-arrays</strong> To quantify this social network in tawny crazy ant colony fragments, we introduced a colony fragment (resident fragment) consisting of 350 workers, 1/8 cm³ of mixed-stage brood, and two queens into the queen chamber of a nest-array. This composition was consistent across all subsequent experiments using the nest array. After 72 hours for the resident fragment to spatially assort, 35 workers were removed from both the peripheral and queen chambers. Workers were identified as Peripheral Chamber Cohort (PCC) or Queen Chamber Cohort (QCC) workers by applying a drop of colored nail polish to their head above the eyes while ants were briefly anesthetized with CO<sub>2</sub>. Pairwise interactions were classified as bump (ants meet and part), antennate (antennal tap for over one second), trophallaxis (regurgitative food sharing), or allogrooming (ants use mouthparts to groom the thorax or abdomen of another ant). These categories were grouped by disease transmission risk (the potential to ingest microsporidian spores): “high-risk" (trophallaxis, allogrooming) and “low-risk" (bump, antennate). Interactions between marked ants were categorized by cohorts: QCC-QCC, QCC-PCC, PCC-PCC. Data were collected across seven nest-arrays. Replicates were defined as the sum of each interaction category observed in a 3-minute focal survey (<em>N</em> = 162 surveys). In a second three-minute survey, we recorded the cohort identity of ants performing tasks. Tasks included corpse removal – carrying dead workers, brood care – touching brood with mouthparts or antennae, and foraging – feeding at either the sucrose tube or cricket. Finally, the number and cohort composition of all groups of ants in the chamber was tallied. Groups were ants in proximity, typically touching, that were not moving or interacting. Replicates were surveys within arenas. Only surveys where specific tasks were observed were included in the analyses: corpse removal (N = 54), brood care (N = 92), foraging (N = 96). <strong>Dispersal of infected and uninfected ants introduced into nest-arrays</strong> To understand how nest structure influences disease spread, we tested whether worker spatial distribution varies with worker source, point of introduction into the nest-array, or worker infection status.  Resident ants (workers, brood, and queens) were introduced into the nest-array queen chamber 72 hours before the introduction of experimental ants. Three types of experimental ants were introduced: 1) nestmates (from the resident, uninfected ant colony fragment), 2) uninfected-aliens, and 3) infected-aliens. Thirty-five experimental ants were introduced into either the queen chamber or the peripheral chamber, making a total of six experimental groups (e.g., nestmates introduced into the peripheral chamber etc.). All experimental ants were color coded with nail polish in the manner previously described.  Typically, two groups were introduced into each array: infected or uninfected alien ants into either the peripheral chamber or the queen chamber, and nestmates into both chambers. In some replicate arrays, nestmates were only introduced into the chamber where alien ants were introduced. Arrays with nestmates introduced into both chambers were used to evaluate the effect of introduction location on the final position of workers, independent of infection status or ant source. The ants were allowed to integrate into the Nest-array for four days, after which the array was disassembled, restricting ants to the chambers where they were located. The final positions of the ants were recorded. To standardize the influence of feeding on ant distribution, fragments were fed a dead cricket 30 minutes before disassembly.  For each array, the response of the experimental group was the average distance of workers from the queen, measured in ‘chamber steps’, with each step represented by a chamber and the connecting nest tube before it. <strong>Behavioral profiles of infected and uninfected workers introduced into nest-arrays</strong> We conducted an additional nest-array experiment, to determine how infection status affects social tasks performed. These followed earlier methods for characterizing social networks of resident ants, with the following exceptions. Introduced, marked ants were from alien colony fragments that were either infected with <em>M. nylanderiae</em> or uninfected. Seventy marked workers (35 infected and 35 uninfected) were introduced into the queen chamber of each array. Data came from 12 nest-arrays, with replicates defined as surveys within each arena. Only surveys in which a specific task was observed were included (corpse removal: N = 112, brood care: N = 240, foraging: N = 238). <strong>Interactions Between Infected and Uninfected Ants</strong> We performed aggression assays to test if infection status affects behaviors during interactions. It involved conflicts between five workers from each nest in non-neutral arenas. Except for the non-neutral arena, the methods followed Roulston et al. 2003. Seventy-two hours before the assay, a 5 cm diameter petri dish lid was placed in an uninfected <em>N. fulva</em> nest box to collect nest odors. A 5 cm plastic ring with 1 cm high walls, painted with Fluon<sup>TM</sup> to prevent ants from climbing out, was placed inside the lid. Five resident ants were placed into the arena via a paintbrush, and five test colony ants were introduced by placing them in glass vials and inverting the vial over the arena. During observation, the nest box was set on a Vivosun<sup>TM</sup> heat mat maintained at 26 °C. Interactions were surveyed at 1, 3, 5, and 7 minutes for 30 seconds, and rated on a seven-point scale; scores of 4 (biting) or greater indicated aggression (adapted from: LeBrun et al. 2019). The average interaction score characterized the arena outcome. Test ants were either nestmates isolated from the resident nest for 24 hours (Isolated Nestmates), uninfected ants from a different local-supercolony (Uninfected-aliens), or workers from an infected colony fragment, from a different local-supercolony (Infected-aliens). Three arenas were run for each pairing, and the mean score from these was used as the response variable. Observers were blind to the origin or infection status of the introduced ants. LeBrun, E.G., K. Ottens, and L.E. Gilbert, *Intracolony transmission of the microsporidian pathogen (Myrmecomorba nylanderiae) and its impact upon the growth of tawny crazy ant (*Nylanderia fulva) <em>colonies</em>. Journal of Applied Entomology, 2017. <strong>142</strong>: p. 114-124.
description: # **Data from: Social immunity in a supercolonial invasive ant: Nest
 structure confers immune function** **Dataset DOI:
 [10.5061/dryad.vx0k6dk5c](https://doi.org/10.5061/dryad.vx0k6dk5c)**
 **Description of the data and file structure** These data were collected
 to assess the role that nest structure plays in determining the efficacy
 of social immune behaviors in preventing the transmission of the pathogen
 *Myrmecomorba nylanderiae* from reaching the core of colony fragments of
 the supercolonial invasive ant species, *Nylanderia fulva.*  **Files and
 variables** * Transmission_Test_Apparatus.xlsx *
 Resident_Social_Network_Tasks.xlsx *
 Resident_Social_Network_-_Interactions.xlsx *
 Worker_Dispersal_in_Apparatus.xlsx * Inf-Uninf_Tasks_in_Apparat.xlsx *
 Inf-Uninf_Interacts_in_Apparatus.xlsx *
 Inf-Uninf_Aggression_in_Non-neutral_Arenas.xlsx **Influence of nest
 structure on infection transmission** File:
 Transmission_Test_Apparatus.xlsx Description: Results of pathogen
 transmission test contrasting transmission in multichambered nest
 apparatus with transmission in simple nest boxes.  Columns: Treatment ID:
 Treatments defined by the infection status and introduction chamber of
 introduced ants.  T1: Uninfected colony fragment housed in a
 multichambered nest apparatus – Infected alien ants introduced into the
 peripheral chamber (IA Peripheral Chamber); T2: Uninfected fragment housed
 in a multichambered nest apparatus - Infected alien ants introduced into
 the queen chamber (IA Queen Chamber); T3: Uninfected fragment housed in a
 simple nest box – Infected alien ants introduced into simple nest box.
 Array vs Box: Whether transmission test occurred in multichambered
 apparatus or simple nest box.  Treatment Name: Treatment name (See
 Treatment ID description).   Collection Site ID: Site where nest was
 collected.  Replicate #: replicate ID.  Apparatus Label: apparatus name. 
 End Sampling (Elapsed days): Last day pupae removed from apparatus or nest
 box and tested for infection. Elapsed Days 1st positive: Elapsed days
 until first pupae tested positive for Mn infection. Elapsed days 2nd
 positive: Elapsed days until second positive test for Mn infection. In
 columns “Elapsed Days 1st positive” and “Elapsed days 2nd positive”, “n/a”
  indicates that no pupae testing positive for the pathogen were recovered.
   **Social networks of resident ants in nest-arrays: tasks and
 interactions** File: Resident_Social_Network_Tasks.xlsx Results of
 tracking the tasks performed by marked worker ants collected from the
 peripheral or queen chamber of the multichamber nest apparatus.   Marked
 ants were immediately returned to the chamber from which they were
 collected. Columns: Apparatus ID: apparatus identifier. Obs Interval:
 observation interval. Cohort ID:  worker cohort carrying out tasks (PC –
 peripheral chamber cohort, QC – queen chamber cohort). Sum of Midden work:
 total midden work tasks observed for cohort Sum of Midden - Carrying Like:
 total midden work tasks observed for cohort carrying dead workers of the
 same cohort Sum of Midden - Carrying Unlike: total midden work tasks
 observed for cohort carrying dead workers of the other cohort Sum of Brood
 Tending: total brood tending tasks observed for cohort Sum of Foraging:
 total foraging tasks observed for cohort Note: Performance of tasks was
 episodic.  Only observation intervals where a task was observed provide
 information on relative task performance.  Observation intervals where no
 relevant tasks were observed entered as “not observed”.  0’s indicate that
 the behavior was observed in that observation interval but no individual
 in the specified cohort was observed doing it.   File:
 Resident_Social_Network_-_Interactions.xlsx Results from tracking the
 types of interactions engaged in by marked worker ants collected from the
 peripheral or queen chamber of the multichamber nest apparatus.   Marked
 ants were immediately returned to the chamber from which they were
 collected. Columns: Array: The multichambered nest array identifier for
 this test.  Obs Interval: The observation interval.  Interaction Category:
 The combined cohort identity of the interacting ants.  Ant cohort identity
 defined from the chamber they were collected from prior to marking (QC-PC:
 queen chamber cohort and peripheral chamber cohort ants interacting,
 PC-PC: peripheral chamber cohort and peripheral chamber cohort ants
 interacting, QC-QC: queen chamber cohort and queen chamber cohort ants
 interacting).   Sum of XXX: The total number of interactions of that type
 observed.  Sum of Risky Interactions (Trophallaxis + Allogrooming): The
 combined sums of interactions deemed likely to transmit pathogen spores
 (trophallaxis and allogrooming).  Sum of Non-Risky  Interactions
 (Bump+Antennate): The combined sum of interactions deemed unlikely to
 transmit pathogen spores (bump and antennate).  Sum of Total Interactions
 Obs: The total number of interaction observed in that interval.
                **Dispersal of infected and uninfected ants introduced into
 nest-arrays** File: Worker_Dispersal_in_Apparatus.xlsx These data provide
 a summary of the average distances in chamber steps from the queen chamber
 that marked workers introduced into the multichambered nest apparatus were
 recovered from the queen and brood of the resident fragment.  Data are the
 average distance all marked workers in that focal category were found from
 the queen. Columns: Apparatus #: The numerical ID of the multichambered
 nest apparatus used.  Focal Worker Category Infection Status (Infected vs
 Uninfected): Infection status of workers to which response variable
 applies (Focal Worker Category). Focal  Worker  Category Origin (Alien vs
 Nestmate)(Alien vs Nestmate): whether introduced ants are from a different
 local-supercolony than the resident fragment or were sourced from the
 resident fragment nest.   Introduction Location - Focal Worker Group:
 whether focal ants were introduced into the queen or peripheral chamber.
 Focal Worker Category (origin- infection status-introduction chamber):
 Complete identifier of focal ants. (Infected-alien workers introduced into
 the Queen Chamber (Inf\_ Alien_Queen Ch.), Infected-alien workers
 introduced into the Peripheral Chamber (Inf_Alien_Peripheral Ch.),
 Uninfected-alien workers introduced into the Queen Chamber
 (Uninf_Alien_Queen Ch.), Unifected-alien workers introduced into the
 Peripheral Chamber (Uninf_Alien_Peripheral Ch.), Nestmates Introduced into
 the Queen Chamber (Uninf_NM_Queen Ch.), Nestmates Introduced into the
 Peripheral Chamber (Uninf_NM_Peripheral Ch.) Avg # of Worker Steps from
 Queen (per worker): Focal Category: Response variable, averaging the
 location of all marked ants in the focal category, the average number of
 chambers they were found from the queen chamber. **Behavioral profiles of
 infected and uninfected workers introduced into multichambered nest
 apparatus: Tasks** File: Inf-Uninf_Tasks_in_Apparat.xlsx Counts of tasks
 observed being performed by infected and uninfected workers in the
 multichambered nest apparatus. Columns: Apparatus ID: apparatus
 identifier. Observation Interval: observation interval. Worker Infection
 Status: Infection status of introduced worker. Sum of Midden work: total
 midden work tasks observed for cohort Sum of Midden - Carrying Infected:
 total midden work tasks observed for cohort carrying dead infected workers
 Sum of Brood Tending: total brood tending tasks observed for cohort Sum of
 Foraging: total foraging tasks observed for cohort Notes: Performance of
 tasks was episodic.  Only observation intervals where a task was observed
 provide information on relative task performance.  Observation intervals
 where no relevant tasks were observed entered as “task not observed”.
 **Behavioral profiles of infected and uninfected workers introduced into
 multichambered nest apparatus: Interactions** File:
 Inf-Uninf_Interacts_in_Apparatus.xlsx Results from multichambered nest
 apparatuses where marked workers of differing infection status were
 introduced.  Data preset counts of interactions observed between workers. 
 Interactions were segregated by the cohort of the interacting ants into
 3-categories: infected worker interacting with another infected worker
 (I-I), infected worker interacting with and uninfected worker (I-U),
 uninfected worker interacting with another uninfected worker (U-U).
 Columns: Apparatus ID: apparatus identifier. Observation Interval:
 observation interval. Interaction Category: The combined cohort identity
 of the interacting ants.  Ant cohort identity defined by infection status
 of natal colony fragment (I-I: marked ant from the infected fragment
 interacting with another marked ant from the infected fragment, U-U:
 marked ant from uninfected fragment interacting with another marked ant
 from the uninfected fragment; I-U: marked ant from the introduced infected
 fragment interacting with an ant from the marked, uninfected fragment.).  
 Sum of XXX: The total number of interactions of that type observed.  High
 Risk  Interactions (So Trophall + So Alogroom): The combined sums of
 interactions deemed likely to transmit pathogen spores: trophallaxis and
 allogrooming. **Aggressive Interactions Between Infected and Uninfected
 Ants** File: Inf-Uninf_Aggression_in_Non-neutral_Arenas.xlsx Results of
 aggression assays conducted in non-neutral arenas to test whether
 infection status alters likelihood of aggression.  Aggression assays were
 conducted in nest boxes housing an uninfected colony fragment (resident
 workers).  Introduced workers were either workers from the same nest that
 had been removed from the nest box 24-hours prior (isolated nest mates),
 uninfected workers collected from a different local-supercolony than the
 resident fragment, or infected workers collected from a different
 local-supercolony than the resident fragment.  Data file presents the
 summary data of 3 arenas confronting workers derived from the same source.
 Columns: Assay Date: Date of test.  Resident Nest  ID - Arena Box: The ID
 of the resident nest which is also the location where the interactions
 occurred (arenas).  Introduced Ants Nest Category: The category for the
 non-resident ants introduced into the arena (Infected Al. – infected
 alien, Iso. Nestmate – isolated nestmates, Uninf Al.- uninfected aliens). 
 Introduced Ants Nest ID: The nest from which introduced ants were
 derived.  Interaction Replicate ID: The ID for the interaction.  
 Interaction - Avg Maximum  Score  (arenas): The average of the maximum
 aggression scores observed across the 3 interactions conducted for each
 Interaction Replicate.   Interaction - Avg  All Scores (arenas): The
 average of all aggression scores observed across the 3 interactions
 conducted for each Interaction Replicate.     **Code/software** Excel