DOI: 10.5061/dryad.1rn8pk110
title: Data and code for: a behavioural and microbiological study of wound care in Camponotus floridanus

description: Open wounds pose a major infection and mortality risk in animals. To
 reduce these risks, many animal species apply antimicrobial compounds on
 their wounds. Ant societies use antimicrobial secretions from the
 metapleural gland to combat pathogens but this gland has been
 lost over evolutionary time in several genera including Camponotus. Using
 behavioral and microbiological experiments, we studied how Camponotus
 floridanus handles infected wounds without the use of antimicrobial
 secretions. When we experimentally injured a worker’s leg at the femur,
 nestmates amputated the injured limb by biting the base (trochanter) of
 the leg until it was severed, thereby significantly increasing survival
 compared to ants that did not receive amputations. However, when the
 experimental injury was more distal (at the tibia), nestmates did not
 amputate the leg and instead directed more wound care to the injury site.
 Experimental amputations also failed to improve survival in ants with
 infected tibia injuries unless the leg was amputated immediately after
 pathogen exposure. Micro CT-scans revealed that the muscles likely
 responsible for leg hemolymph circulation are predominantly in the femur.
 Thus, it is likely that femur injuries, by attenuating hemolymph flow,
 provide sufficient time for workers to perform amputations before pathogen
 spread. Overall, this study provides the first example of the use of
 amputations to treat infected individuals in a non-human animal and
 demonstrates that ants can adapt their type of treatment depending on the
 location of wounds.
description: Data includes: 1) Behavioral
 ethograms 2) Survival data 3)
 Bacterial qPCR 4) R-Code for analyses of datasets
 1-3 5) PDF with R-code on the Bayesian Model in Fig.
 S1D 6) PDF with R-code for Figures 1-3
description: # **Raw Excel Data and Code**
 [https://doi.org/10.5061/dryad.1rn8pk110](https://doi.org/10.5061/dryad.1rn8pk110) **2020_tracking_floridanus_control_femur.csv** Behavioural Ethogram Data for Figure 1 -       ID: individual ID of focal ant -       Trophalaxis: amount of seconds performing trophalaxis -       Allogrooming amount of seconds focal individual received allogrooming -       Percent_allo: percentage of 10 min interval in which allogrooming was received -       Wound_licking: amount of seconds focal individual received wound care -       Percent_wound: percentage of 10 min interval in which wound care was received -       Woundlicker: number of ants performing wound care in 10 min interval -       Amputation: amount of seconds focal individual received amputation behaviour on trochanter -       Percent_amp: percentage of 10 min interval in which amputation behaviour was received -       Time_point: 10 min interval of observation -       Time: time intervals as continous variable -       Wounding: wound type numerical categories (1=injured; 2= amputated, 3=healthy) -       State: wound type factor categories (injured, amputated, healthy) -       Colony: Ant colony ID used for the experiment **2021_tracking_floridanus_tibia_femur.csv** Behavioural Ethogram Data for Figure 2 -       ID: individual ID of focal ant -       Trophalaxis: amount of seconds performing trophalaxis -       Allogrooming: amount of seconds focal individual received allogrooming -       Percent_allo: percentage of 10 min interval in which allogrooming was received -       Allogroomer: number of ants performing allogrooming in 10 min interval -       Wound_licking: amount of seconds focal individual received wound care -       Percent_wound: percentage of 10 min interval in which wound care was received -       Woundlicker: number of ants performing wound care in 10 min interval -       Amputation: amount of seconds focal individual received amputation behaviour on trochanter -       Percent_amp: percentage of 10 min interval in which amputation behaviour was received -       Amputater: number of ants performing amputation in 10 min interval -       Time_point: 10 min interval of observation -       Time: time intervals as continous variable -       Wounding: wound type numerical categories (1=femur; 2= tibia) -       Wounding1: wound type factor categories (femur, tibia) -       State: wound type including amputations (femur, tibia, amputated) -       Colony: Ant colony ID used for the experiment **2021_isolation_experiment_TIBIA5_colony.csv** Survival Experiment Data for Figure 3A -       ID: individual ID of focal ant -       Colony: Ant colony ID used for the experiment -       Treatments: Type of injury manipulation (Healthy, Sterile or infected wound, injury at tibia or femur, inside the nest or in isolation) -       Time: time of death/end of experiment -       Survival: binary code for alive/dead at timepoint (1=alive, 2=dead) **2_Dany_MainExp_qPCRdata.csv** qPCR data for bacterial quantification for Figure 3B Sample_ID and ID: Individual ID of the analysed sample Target: Pseudomonas 16S genes targetted for qPCR analyses Ct and SD values for the individuals (see method description below for calculations) 28S_Cq and dCq calculated dCq values (see method description below for calculations) Treatment: Type of wound manipulation (tibia or femur, infected or sterile, amputated or not) State: State of the wound (sterile, infected amputated) Injury: Location of the wound (tibia or femur) Colony: Ant colony ID used for the experiment **amputation.csv** Sample Size: Number of ants used for each group Wound location: Location of the wound (tibia or femur) Amputation: Number of ants whose wound was amputated after 24h (0=no amputation; 1=amputation) Wound type: State of the wound (infected or sterile) Colony: Ant colony ID used for the experiments ## Description of the data and file structure Behavioural csv sheets (*tracking*) for Figure 1E and 2 include behavioural observations binned in 10 minute intervals. Detailed explanation on the terminology and experimental design can be found in the Methods section below. Survival csv sheet (*isolation*) for Figure 3AB includes survival data structured for analysis with the survminer package in R. Detailed explanation on the terminology and experimental design can be found in the Methods section below. qPCRdata csv sheet (*_qPCRdata*) for Figure 3CD includes CT valuesof bacterial abundance inside the hemolymph for each individual. Detailed explanation on the terminology and experimental design can be found in the Methods section below. All the data can be analysed and illustrated with the provided R-code (R-code Figures 1-3 and Bayesian model). **EXPERIMENTAL MODEL AND SUBJECT DETAILS** ***Camponotus floridanus* colonies** Colonies were reared from queens collected in 2017 (Florida, United States) and kept at the University of Lausanne in a climatized room at 26 °C with 65% humidity and a 12h day/night cycle. Although workers of *C. floridanus* show a continuous size distribution, there are two clear recognizable castes, minors (length = 5.5-7mm, head width &lt; 1.5mm) and majors (length = 8-10mm, head width &gt; 2.7mm). 16 Ants were kept for one week in experimental boxes (17.5x23cm) for acclimatization before the experiments. We used only minor workers in the experiments. **METHOD DETAILS** **Wound care behavior of *C. floridanus*** Focal individuals were color tagged with acrylic paint two days before each experiment. In a first experiment, we compared the wound care behaviors towards ants with a sterile femur injury (primary wound) and their subsequent injury after amputation of the injured leg by nestmates (secondary wound). In each of four sub-colonies (each containing 47 foragers, 50 nurses and 3 majors but no brood or queen), we placed two ants whose right hind legs were cut at the center of the femur (Figure 1E). All experimental injuries were done with a sterile Dowel-scissor (Fine Science Tools). Before cutting the leg, the focal individuals were cooled for 2 minutes by placing them in a glass container surrounded by ice. This allowed easier manipulation and precision during the cut, which was always located at the center of the tibia or femur. Each sub-colony was placed in a climate box and filmed with an infrared camera for 24 hours after injury. We then quantified the first three hours after injury and the first three hours after amputation. In a second experiment, we compared the wound care behaviors toward injured nestmates with either a sterile wound at the level of the femur or tibia. Ants’ right hind legs were cut either at the center of the femur (*N*=10) or center of the tibia (*N*=10). Two femur-injured and two tibia-injured individuals were then returned in each of five naive sub-colonies containing 95 foragers, 100 nurses and 5 majors (Figure 2). There were thus two femur- and two tibia-injured ants inside each sub-colony at the same time. Because one femur- and one tibia-injured ant lost the color ID, sample sizes are *N*=9 per treatment. Sub-colonies were filmed with an infrared camera in climate boxes for one hour before the treatments and for the subsequent 6 hours. To maintain the ratio of healthy to injured ants the same across experiments, we had to double the number of individuals in each sub-colony in the second experiment (from 100 ants in Figure 1, to 200 ants in Figure 2). Both experiments were conducted in November and December 2020. Behavioral analyses of the videos were performed using VLC media player v. 3.0.11-win64 with the help of the add-on Zoomit. Wound care behaviors were classified into two categories: (1) “wound care” when nestmates did groom/lick the subject at the wound and (2) “amputation” when nestmates bit the injured leg on the trochanter. Behaviors were quantified in 10 min sections for the 6 hours following the injury.  **Survival of injured ants** We conducted four treatments to test the effect of wound care and amputation on the survival of individuals injured either at the center of the femur or tibia. In the first treatment, a sterile PBS solution was applied on the wound and the individuals were isolated. The second treatment was identical, but the PBS solution contained *P. aeruginosa*. In the third treatment, we also applied a PBS solution containing *P. aeruginosa* but returned the wounded individual to a sub-colony comprising 200 workers. Finally, the fourth treatment was identical to treatment 2, but we experimentally amputated the injured leg with sterile microscissors at the level of the trochanter one hour after injury. The four treatments were conducted both for ants injured at the level of the femur or tibia. Six ants from each of the six sub-colonies were used per treatment, making a total of 36 ants per treatment, 24 of which were used for the survival analysis (*N*=24) and 12 were collected after 35h for the qPCR analyses (outlined in the next section; *N*=12). In all treatments, ants had access to food (honey water) and water. For treatments 2-4, injuries were infected by exposing the injury to a 10 μL solution of gram-negative bacteria (*Pseudomonas aeruginosa*) diluted in sterile phosphate-buffered saline (PBS; optical density = 0.005; approx. 105 bacteria in 10μL of PBS) following the same protocol and pathogen strain of Frank et al. 2023. 9 For treatment 1 ants had their wound exposed to a sterile solution of PBS. Manipulated ants were checked once per hour for the next 48 hours and once every two hours from 48 to 72 hours. To assess the impact of the delay between pathogen exposure and experimental amputation on survival for femur- and tibia-injured ants, we repeated the same survival experiment as before but with five different amputation times: 0, 5, 10, 30 and 60 minutes after pathogen exposure (Figure S4). Each treatment included 12 replicates, each from a different colony. One replicate had to be removed due to a humidity malfunction in one of the systems and four individuals were removed from the analysis because they died before the survival recordings began due to handling mistakes. This second survival experiment was conducted in December 2023. **Bacterial quantification** To compare the proliferation of bacteria in infected ants kept in isolation or inside sub-colonies, we performed qPCR analyses 35 hours after manipulation on a third of the individuals from the survival experiment (*N*=12 for each of the 8 treatments; Figure 3CD). The gasters of the focal individuals were removed to reduce potential noise from the gut bacteria. DNA was extracted from the bodies by snap-freezing them in liquid nitrogen for better homogenization with a Precellys Evolution homogenizer (Bertin Technologies) at 6500 rpm for 2 x 30 s using a pool of zirconium ceramic beads. We then added 180 µl of ATL buffer and 20 µl of proteinase K (20 mg ml−1) and digested these homogenates at 56 °C overnight, after which DNA was extracted using a Qiagen BioSprint 96 robot with the BioSprint DNA Blood Kit following the manufacturer’s instructions. Bacterial loads were quantified with a QuantStudio qPCR instrument (Applied Biosystems) using thermal cycling conditions as recommended for SYBR® Select Master Mix and using the protocols published in Kešnerová et al. 17 To calculate *P. aeruginosa* bacterial loads, we used primers Ps-16S-fw 5’-GTAGATATAGGAAGGAACACCAG-3’ and Ps-16S-rv 5’-GGTATCTAATCCTGTTTGCTCC-3’, which were originally developed for Frank et al. 2023, 9 and for normalization we targeted the *C. fellah* 28S gene with primers 28S-fw 5’-CTGCTCGGCGGTACGCG-3’ and 28S-rv 5’-ACCGGGGACGGCGCAAA-3’. *P. aeruginosa* 16S rRNA gene copy numbers (target gene) were expressed relatively to *C. fellah* 28S rRNA reference gene copy numbers based on the Pfaffl method 18 using the equation: ΔCt = (Etarget)ΔCt, target (calibrator - test)/(Eref)ΔCt, ref (calibrator - test), where Etarget is the amplification efficiency (calculated based on a 10X serial dilution) of the Pseudomonas 16S rRNA gene (Etarget = 2), Eref is the amplification efficiency of the 28S *C. fellah* reference gene (Eref = 1.827); ΔCt, ref (calibrator - test) is the Ct of the reference gene in a sample used as calibrator (sample 13) minus the Ct of the reference gene in the test sample; ΔCt target (calibrator - test) is the Ct of the target gene in the calibrator minus the Ct of the target gene in the test sample.