DOI: 10.5061/dryad.w6m905r28
title: Data from: Better safe than sorry: Leg amputations as a prophylactic wound care behaviour in carpenter ants

description: Animals often sustain injuries that are susceptible to lethal infections.
 In social insects, wound care behaviours have evolved to reduce these
 risks. But the limits of wound care behaviours remain unclear. Here we
 investigated the wound care behaviours of the ant Camponotus maculatus.
 Our findings show that amputation of legs infected with Pseudomonas
 aeruginosa significantly reduced mortality. However, nestmates do not
 differentiate between infected and sterile injuries, providing the same
 treatments regardless of the infection. Even though we show that early
 amputation correlates with higher survival rates, nestmates amputate
 indiscriminately on legs with fresh or old wounds. Additionally, cuticular
 hydrocarbon profiles differed between ants with infected or sterile wounds
 only 24 hours post-injury, a timepoint when amputations are no longer
 effective. We propose that C. maculatus workers perform prophylactic
 amputations regardless of injury state or age. This is in sharp contrast
 to previous studies, which showed clear capabilities to treat infected
 wounds differently in ants using antimicrobial compounds. This work,
 therefore, shows the limits of wound care behaviours in social insects,
 allowing us to better understand the evolutionary drivers of this unique
 behaviour.
description: Experimental
 designs: <strong>Wound care behavioural
 experiments</strong> To compare the wound care
 behaviours towards infected and sterile-injured ants, we created 3
 sub-colonies between November 8 and 21, 2022. Six previously marked ants
 were collected per sub-colony, and all of them were wounded at the centre
 of the right hind femur using sterile scissors. Afterwards, half were
 exposed to a 10 μL pathogenic solution by immersing their injury in a
 solution containing approximately. 10<sup>5</sup> <em>P.
 aeruginosa</em> bacteria (0.01 OD) for 2 seconds. In the control
 group, the wound was exposed to sterile PBS for 2 seconds. All the workers
 were then returned to their respective sub-colonies for observations (N=9
 per group). The experiment was also repeated with a
 higher infection dose, infecting ants with a ten times more concentrated
 <em>P. aeruginosa</em> solution (0.1 OD, approx.
 10<sup>6</sup> <em>P. aeruginosa</em> bacteria).
 Observations followed the same procedure as above (N=9 per group). While
 these sample sizes are relatively small (reflecting the substantial effort
 of continuous manual ethogram annotation per individual), they were
 sufficient to identify clear overall patterns that support our
 conclusions. We therefore do not believe that larger sample sizes would
 have altered the main findings. However, as with any study based on
 limited replication, effect sizes near the margins should be interpreted
 with caution, and future work with larger datasets may be able to refine
 or detect subtler trends with greater statistical power.
 Treated ants were observed in the nest for two weeks, recording
 the number of nearby ants, the treated worker’s condition (alive or dead),
 and overall colony mortality. Deceased ants were removed and inspected for
 limb amputation. Data on surviving ants and amputations were recorded
 every hour for the first 24 hours, every two hours for the remainder of
 the first week, and twice a day during the second week. Ant behaviour
 inside the nest was monitored using a Panasonic HC-X1000 4K video camera
 recorder mounted on top of the nest box. We analysed the first 6 hours of
 video recordings for each trial using VLC media player 3.0.16 Vetinari. We
 distinguished the following behaviours: (I) Wound care (injury is cleaned
 by another worker), (II) allogrooming (cleaning the injured worker), and
 (III) amputation (biting on the trochanter by a nestmate).
 <strong>Survival experiments</strong>
 To confirm the role of amputation in reducing mortality rates
 associated with injury and wound infection, we compared the survival of
 injured ants with or without amputation and infection. A total of 80
 previously uninjured workers (from 4 different colonies) were divided into
 4 treatments. The first group, labelled “Healthy,” served as a negative
 control and received no treatment (N=20). In the second group, “Sterile,”
 the right hind leg was severed at the centre of the femur using sterile
 scissors, and the wound was soaked in sterile PBS for 5 seconds (N=20).
 Workers in the “Infected” group were treated in the same manner as the
 “Sterile” group, but their wounds were soaked in a 0.01 OD <em>P.
 aeruginosa</em> solution for 2 seconds (N=20). The “Infected +
 Amputated” group underwent the same initial treatment as the “Infected”
 group, but after one hour, the leg was manually amputated at the
 trochanter using sterile scissors to simulate amputations performed by
 nestmates (N=20). All workers were kept in isolation chambers and provided
 <em>ad libitum</em> honey water and water. Isolation
 experiments were performed in parallel with a social experiment where 60
 workers were collected from the same 4 colonies and received the same
 treatments as described above: (1) “Healthy”, (2) “Sterile”, and (3)
 “Infected”. All the workers were then put back into their respective
 sub-colonies (N=20 per group). The isolation and colony survival
 experiments were repeated, using a 0.1 OD <em>P.
 aeruginosa</em> solution (N=20 per group). Workers were monitored
 once per hour for 6 days (144h), and the time of death was
 recorded. To determine the time window during which
 amputations by nestmates remained effective in reducing infection
 mortality, we manually amputated the infected leg of injured ants kept in
 isolation at different time points. 10 workers (N=10) were collected per
 treatment group from four colonies. After manually injuring the femur and
 exposing the wound to a <em>P. aeruginosa</em> solution (0.1
 OD), we amputated the infected leg at 0-, 6-, 12-, or 24-hours
 post-exposure. Mortality was quantified for the first 6 days
 post-exposure. To investigate whether the duration ants
 carried an infected or sterile injury influenced the frequency of
 amputations by nestmates, we introduced injured nestmates at different
 timepoints after injury and/or infection into sub-colonies. Individual
 workers were collected from each of the 4 colonies and received one of
 three treatments: (1) “Healthy”, (2) “Sterile”, or (3) “Infected”. Half of
 the workers were immediately returned to the nest after manipulation, and
 the other half were held in isolation chambers with water and honey water
 provided <em>ad libitum</em> for 24 hours before being
 reintroduced to the nest (N=10 per group and time-point).
 <strong>Cuticular hydrocarbon
 analysis</strong> A total of 70 workers were
 collected from one colony (N=70). Of these, 20 received a <em>P.
 aeruginosa</em> infected injury (OD 0.1), 20 received a sterile
 injury, and the remaining 30 did not receive any additional treatment as
 control (“healthy”). 10 individuals per group were frozen at -20°C, 2h,
 and 24h after manipulation, in addition to 10 healthy ants at 0h. Before
 freezing, the ants were kept alone in isolation in sterile petri dishes
 containing ad libitum water and honey water.
description: # Data from: Better safe than sorry: Leg amputations as a prophylactic
 wound care behaviour in carpenter ants Dataset DOI:
 [10.5061/dryad.w6m905r28](10.5061/dryad.w6m905r28) ## Description of the
 data and file structure The zip file Analyses.zip contains folders divided
 into 4 analyses sections (analyses_chc_infection;
 analyses_efficiency_amputations; analyses_ethograms;
 analyses_time_amputations). Data analysis was conducted using R and
 RStudio. The zip file Data.zip contains 4 folders with the respective raw
 data (data_chc; data_efficiency_amputations; data_ethograms;
 data_time_amputations). Missing data code: NA Each analysis folder has a
 corresponding .Rmd file with the code necessary to recreate all the
 figures and analyses from the study using the raw data csv files provided
 in the respective data folder. The .Rmd files are also annotated to
 explain each line of code. #### File: Analyses.zip
 **Description:** includes all the code to reproduce all the results of the
 study (including figures). #### File: Data.zip **Description:** includes
 all the raw data to reproduce all the results of the study. For data_chc
 there are 2 csv files: **Mastertable_Camponotus_Final.csv** * Compound_ID:
 refers to our manually identified compound using Nist library and MS
 spectra * Retention_Time: Retention time in minutes of our Gas
 Chromatographic run * Retention_Index: The universal index calculated
 using retention time of the respective alkanes. * Compound: Compound class
 the respective compound belongs to * Chainlength: The number of carbon
 atoms in the compound * Subsequent lines refer to the individual samples
 with G=Healthy; I=Infected; S=Sterile; 0h= zero hours after manipulation;
 2h= 2 hours after manipulation; 24h = 24 hours after manipulation. The
 last number refers to the sample repeat. e.g. the 7th healthy sample at
 timepoint 2h is called: G2h_7. **Metadata_Camponotus.csv** This file
 contains the metadata necessary for analysis. * ID= refers to the
 identification of the sample. Same code as used above but preceeded by
 Camponotus\_ * Treatment= refers to healthy (uninjured), sterile or
 infected wounds. * Time (h) = refers to how many hours after treatment the
 CHC profile was extracted (0, 2 or 24h) For data_efficiency_amputations
 all csv files follow the same nomenclature.
 **Mortality_Ants_0.1OD_144h.csv** refers to an experiment in which wounds
 were infected with a concentration of 0.1OD of pseudomonas bacteria, while
 **Mortality_Ants_0.01OD_144h.csv** refers to an experiment in which a ten
 times lower pathogen dose was used. * time= time the individual was last
 observed (dead or alive) * event= signals if at that time the individual
 was dead (2) or alive (1) * treatment= same meaning as above. Healthy=
 uninjured, infected (signals either if the sample was infected with a high
 or low pathogen dose, see csv file name either \_0.1OD or \_0.01OD);
 sterile= injured ant without infection; amputated= infected ant whose leg
 was amputated after set amount of time. * location= signals if the ants
 were in their colonies (colony) or kept alone in isolation. * colony=
 states the mother colony of the trialed individual * amputated= states if
 the individual was amputated inside the colony. * notes= include observer
 additional notes. ertrunken= individual drowned in the provided food in
 the isolation chambers. For data_ethograms the behavioral ethograms were
 quantified. **data_ethograms_infected_sterile.csv** * Datum= The date in
 which the experiment was conducted * ID= the individual ID of the tested
 individual * Versuch= the number of the experiment * Kolonie = the colony
 used for the experiment * Farbcode = the color used to mark the individual
 (with y=yellow, b=blue, g= green, ry= red and yellow, rb= red and blue,
 rg= red and green) * Behandlung = Treatment as outlined above, with
 infiziert= infected, steril= sterile. * Wundtyp= if the wound was
 amputated or not by nestmates. verletzt= injured by us. amputiert= leg
 amputated by nestmates * Zeit (absolut) = Time intervall of the ethogram
 (in steps of 10 minutes) * Zeit(wunde) = Total time the original wound or
 amputation was present * Intervall = Actual time the individual could be
 observed * Ant(n)= number of antennations by others * Ant(t)=duration of
 antennations by others * Troph(n) =number of trophalaxis by others *
 Troph(t) = duration of trophalaxis by others * BebbB(n) = number of
 begging behaviour by others * BeggB(t) = duration of begging by others *
 SelfG(n) = number of selfgrooming by others * SelfG(t) = duration of
 selfgrooming by others * AlloG(n) = number of allogrooming by others *
 AlloG(t) = duration of allogrooming by others * WundB(n) = number of
 woundcare by others * WundB(t) = duration of woundcare by others * AmpV(n)
 = number of amputation attempts by others * AmpV(t) = duration of
 amputation behaviour by others * Amputation = amputation time
 **isolation_infected_sterile_144h.csv** * ID= individual ID * Behandlung=
 Treatment for that individual I=infected; s=sterile * Zeit= Time of last
 observation (dead or alive) * Überleben= Signaling dead (1) or alive (0)
 **probability_amputations.csv** * Date= Refers to the date of the
 experiment * ID= individual id * Colony= mother colony of individual *
 Treatment= Infected or Sterile wound * Time_2= Time in hh:mm:ss * Time=
 Time in minutes * Amputation= Time of amputation. 0= no amputation, 1=
 amputated **time_to_amputation.csv** * Date= Refers to the date of the
 experiment * ID= individual id * Colony= mother colony of individual *
 Treatment= Infected or Sterile wound * Time_to_amputation= Time of
 amputation in hh:mm:ss * Seconds= Time to amputation in seconds *
 Amputated_during_6h= Signifies if amputation occured within the first 6
 hours: 1=yes, 0= no The last folder data_time_amputations contains 3 csv
 files **amputations_after_24h.csv** * Colony= Mother colony of individual
 * Time = Time after wounding the individual is introduced in the colony *
 Treatment= If wound is sterile or infected * Amputated= how many
 individuals were amputated * Total= how many individuals were placed in
 the colony * Time_amp_checked= time amputation was first discovered (in
 hours) **amputations_time_fixed.csv** * Colony= Mother colony of
 individual * ID= Individual ID of sample * Hour= Time of death *
 Treatment= Infected or sterile wound * Leg = Treatment on the leg by us
 after injury. Injured= not amputated. Amptuated= amputated by us after a
 set amount of time (see next column) * Time= Time of artificial amputation
 (in hours) * Condition = Dead (1) or Alive (0). ## Code/software For
 statistical analyses and graphical illustrations, we used the statistical
 software R v4.3.2 (R Core Team 2025) with the user interface RStudio
 2023.06.0+421 (Rstudio Team 2020) and the R package ggplot2 v.3.3.5
 (Wickham 2016). To investigate behavioural differences between workers
 with infected and sterile wounds, we modelled wound care and allogrooming
 as binary response variables using Hierarchical Generalised Additive
 Models (HGAM) with a binomial family. A nested random effect with a
 smoother was included for each individual within a colony, along with a
 smoother interaction between time and wound condition. To identify time
 intervals with significant differences, we conducted post-hoc contrasts on
 the probability of these behaviours between infected and sterile ants.
 Given that wound care often occurs in short, intermittent bursts, we
 considered wound care to be present if it was observed for at least
 one-quarter of the observation interval (2.5 minutes). In contrast,
 allogrooming, which is more frequent during ant interactions, was
 considered present if it occurred for at least half of the observation
 interval (5.0 minutes). The conclusions remained robust regardless of
 variations in these criteria. Similarly, to determine when amputations
 were most likely to occur, we recorded the time at which amputated ants
 were first observed. Binary records per treatment were analysed using an
 HGAM model, as applied for allogrooming and wound care. Model assumptions
 were assessed using the *DHARMa* v.0.4.7 (Hartig 2024) and *mgcv* v.1.9.1
 (Wood 2017) packages. To determine whether ants with infected wounds
 undergo amputation sooner than those with sterile wounds, we used
 Generalised Linear Models (GLMs) to model the time to amputation as a
 function of wound condition with the package *lme4* v1.1.33 (Bates et al.
 2015). Colony ID was included as a random effect to account for
 colony-level variation. To evaluate the impact of amputation on mortality
 at low and high pathogen concentrations, we fitted separate Mixed Effects
 Cox Models to analyse censored survival data for each experiment. The
 models included treatment (healthy, sterile, infected, infected +
 amputated) and environment (colony, isolated) as additive effects, with
 Colony ID as a random effect to account for colony-level variation. Model
 fitting was performed using the *coxme* v.2.2.22 (Therneau 2024). To
 assess at which time the amputations are no longer efficient in reducing
 mortality caused by infections, we fitted a Mixed Effects Cox Model for
 the censoring data on the survival of amputated ants at different times.
 Colony ID was included as a random effect. Furthermore, to validate that
 the mortality of workers was caused by the infections, we compared the
 survival of workers with an infected and sterile wound by cumulative
 Kaplan-Meier curves with the packages *Surv* v.3.5.5 and *survminer* v.0.5
 (Kassambara et al. 2024). To identify differences in the mortality of
 specific groups, we performed pairwise comparisons on estimated marginal
 means. We controlled for false discovery rates on multiple comparisons by
 Tukey adjustments using the package *emmeans* v.1.11.1 (Lenth 2025). CHC
 analyses were performed using the packages ggtext, gplots, GCalignR,
 ggdist, ggside and analyzeGC. Chromatographic data was aligned in R using
 the package GcalignR v.1.0.7 (Ottensmann et al. 2018). Subsequently, all
 compounds with a relative abundance below 0.1% (too small to identify) or
 less than 50% occurrence within all groups were excluded, so as to focus
 the CHC analyses to differences across groups of interest as done in
 previous studies (Alciatore et al. 2021, Yusuf et al. 2020). The final
 compounds were then identified, and a retention index was calculated based
 on Carlson et al. (1998). To visualise dissimilarities between wound types
 over time, we calculated the Bray-Curtis dissimilarities indices for the
 relative abundance data between CHC samples. We further visualise these
 dissimilarities by performing non-metric multidimensional scaling (NMDS).
 To test whether ants sustaining infected wounds show a distinct CHC
 profile, we assessed differences between groups over time by performing a
 multivariate analysis of variance (PERMANOVA) with 9999 permutations.
 These analyses were performed with the package veganv.2.6-10 (Oksanen et
 al. 2025).