Better safe than sorry: Leg amputations as a prophylactic wound care behaviour in carpenter ants

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.

Methods

Experimental designs:
Wound care behavioural experiments

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. 105 P. aeruginosa 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 P. aeruginosa solution (0.1 OD, approx. 106 P. aeruginosa 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).

Survival experiments

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 P. aeruginosa 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 ad libitum 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 P. aeruginosa 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 P. aeruginosa 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 ad libitum for 24 hours before being reintroduced to the nest (N=10 per group and time-point).

Cuticular hydrocarbon analysis

A total of 70 workers were collected from one colony (N=70). Of these, 20 received a P. aeruginosa 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.