Targeted treatment of injured nestmates with antimicrobial compounds in an ant society

Abstract

Infected wounds pose a major mortality risk in animals. Injuries are common in the ant Megaponera analis, which raids pugnacious prey. Here we show that M. analis can determine when wounds are infected and treat them accordingly. By applying a variety of antimicrobial compounds and proteins secreted from the metapleural gland to infected wounds, workers reduce the mortality of infected individuals by 90%. Chemical analyses showed that wound infection is associated with specific changes in the cuticular hydrocarbon profile, thereby likely allowing nestmates to diagnose the infection state of injured individuals and apply the appropriate antimicrobial treatment. This study demonstrates that M. analis ant societies use antimicrobial compounds produced in the metapleural glands to treat infected wounds and reduce nestmate mortality.

Full text

Article https://doi.org/10.1038/s41467-023-43885-w
Targeted treatment of injured nestmates
with antimicrobial compounds in an ant
society
Erik.T.Frank
1,2
,LucieKesner
3
, Joanito Liberti
1,3
,QuentinHelleu
1,4
,
Adria C. LeBoeuf
5
,AndreiDascalu
1
, Douglas B. Sponsler
2
,FumikaAzuma
6
,
Evan P. Economo
6,7
, Patrice Waridel
8
,PhilippEngel
3
, Thomas Schmitt
2
&
Laurent Keller
1
Infected wounds pose a major mortality risk in animals. Injuries are common
in the ant Megaponera analis, which raids pugnacious prey. Here we show
that M. analis can determine when wounds are infected and treat them
accordingly. By applying a variety of antimicrobial compounds and proteins
secreted from the metapleural gland to infected wounds, workers reduce the
mortality of infected individuals by 90%. Chemical analyses showed that
wound infection is associated with specific changes in the cuticular hydro-
carbon profile, thereby likely allowing nestmates to diagnose the infection
state of injured individuals and apply the appropriate antimicrobial treat-
ment. This study demonstrates that M. analis ant societies use antimicrobial
compounds produced in the metapleural glands to treat infected wounds
and reduce nestmate mortality.
Infections are a major mortality risk in animals1,2, with the risk of
transmission of contagious pathogens being particularly life-
threatening in group living animals3. This has led to a suite of
pathogen-induced changes in social interactions, like socialdistancing,
sickness cues, and medical care4–6. In injured individuals, the major
barrier against infections (the cuticle or epidermis) is damaged and
therefore provides an easy entry point for life-threatening infections7.
Recently, several mammals have been shown to lick wounds to apply
antiseptic saliva1,5.However,theefficacy of these behaviors remains
largely unknown and occur irrespective of the state of the wound.
In social insects, interactions to combat pathogens range from
preventive measures like nest disinfection or allogrooming to mor-
ibund individuals leaving the nest to die in isolation or the destructive
disinfection of their infected brood3,8–10. But if and how social insect
colonies care for injured individuals that were exposed to pathogens
remains poorly understood. Workers of the predatory ant Megaponera
analis have been shown to care for the injuries of nestmates2,7,which
are common because this antfeeds exclusively on pugnacious termite
species11. As many as 22% of the foragers engaging in raids attacking
termites have one or two missing legs2. Injured workers are carried
back to the nest where other workers treat their wounds, by licking and
grooming the wound during the first three hours after injury7.When
the wounds of injured workers are not treated by nestmates, 90% of
the injured workers die within 24 h after injury7, but the mechanisms
behind these treatments are unknown. The aim of this study is there-
fore to identify the cause of death in injured individuals and the
potential mechanisms involved in the detection and treatment of
injuries.
Received: 26 May 2023
Accepted: 20 November 2023
Check for updates
1
Department of Ecology and Evolution, Biophore, University of Lausanne, CH-1015 Lausanne, Switzerland.
2
Department of Animal Ecology and Tropical
Biology, Biocenter, University of Würzburg, D-97074 Würzburg, Germany.
3
Department of Fundamental Microbiology, Biophore, University of Lausanne,
CH-1015 Lausanne, Switzerland.
4
Structure et Instabilité des Génomes, Muséum National d’Histoire Naturelle, CNRS UMR 7196, INSERM U1154, 43 rue
Cuvier, F-75005 Paris, France.
5
Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland.
6
Biodiversity and Biocomplexity Unit,
Okinawa Institute of Science and Technology Graduate University, Onna 904-0495, Japan.
7
Radcliffe Institute for Advanced Study, Harvard University,
Cambridge 02138, USA.
8
Protein Analysis Facility, Génopode, University of Lausanne, CH-1015 Lausanne, Switzerland.
e-mail: erik.frank@uni-wuerzburg.de;laurentkeller01@gmail.com
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Here we show that the gram-negative bacterium Pseudomonas
aeruginosa can cause lethal infections in injured M. analis workers. We
found that infected wounds are treated more often than sterile
wounds and that this correlates with changes in the cuticular hydro-
carbon profile of infected individuals. We describe the use of the
metapleural gland to treat infected wounds and quantify its content,
identifying 112 chemical compounds and 41 proteins in the gland’s
secretion, half of which have antimicrobial or wound healing proper-
ties. Overall, this study demonstrates that the targeted use of anti-
microbials to treat infected wounds is effective in decreasing mortality
in injured individuals.
Results and discussion
Pathogen identification and mortality cause
To investigate whether the high mortality of injured individuals is
due to infection by pathogens, we collected soil from the natural
environment and applied it to the wounds of experimentally injured
workers (i.e., a sterile cut in the middle of the femur on the hind leg
of an otherwise healthy ant). After 2 h, injured ants exposed to the
soil (hereafter referred to as “infected ants”) had ten times higher
bacterial loads in the thorax than similarly injured individuals
exposed to sterile phosphate-buffered saline (PBS, hereafter referred
to as “sterile ants”; least square means: degrees of freedom (DF) = 35;
t=−3.08; P= 0.01; Fig. 1a and Supplementary Table 1). After 11 h,
bacterial load further increased in infected ants (least square means:
DF = 35; t=−3.66; P= 0.003), while there was no such increase in
sterile ants (least square means: DF = 35; t=−1.54; P= 0.26; Fig. 1a). As
a result, after 11 h the bacterial load was 100 times higher in infected
than sterile ants (least square means: DF= 35; t=−5.21; P< 0.001;
Fig. 1a). A microbiome analysis further revealed major differences in
bacterial species composition and abundance between the two
groups of ants (ADONIS: F
(1,39)
= 17.45; R2= 0.31; P< 0.001; Fig. 1band
Supplementary Fig. 1a, b), with three potentially pathogenic bacterial
genera increasing in absolute abundance in the thorax of infected
ants at both time-points: Klebsiella,Pseudomonas and Burkholderia
(Fig. 1b and Supplementary Table 2).
To investigate if the differences in bacterial abundance and
composition affect survival, we either placed infected and sterile ants
in their colony or kept them in isolation. The mortality after 36h was
much lower for infected ants kept with their nestmates (22%) than for
infected ants kept in isolation (90%, least square means: DF = 154;
Z=−2.759; P= 0.02; Fig. 1c). By contrast, there was no significant dif-
ferencebetween the mortality of sterile ants kept with their nestmates
or in isolation (least square means: DF =154; Z=1.04;P=0.89;Fig.1c).
The mortality of infected ants was also not significantly different than
the mortality ofsterile ants when these individuals were kept with their
nestmates (least square means: DF = 154; Z=−0.630; P= 1; Fig. 1c).
Overall, these data demonstrate that M. analis workers are capable of
effectively treating wounds that have been exposed to soil pathogens
through social interactions.
By culturing the soil medium on agar plates, we were able to
isolate three potential pathogens (the endosymbiotic bacterium Bur-
kholderia sp. and its fungal host Rhizopus microsporus, and the bac-
terium Pseudomonas aeruginosa,Fig.1b and Supplementary Fig. 1c, d).
While the application of B.sp.andR. microsporus (separately or
together) on wounds did not significantly decrease survival (Supple-
mentaryFigs. 2b and 3), the application of P. aeruginosa (OD = 0.05), a
bacterium widespread in various environments12, caused a mortality of
93% within 36 h (Fig. 2a). Since the treatment with only P. aeruginosa
showed a similar mortality as the treatment with all soil pathogens
(90%, Supplementary Fig. 3), we only used P. aeruginosa in subsequent
infection assays to better control pathogen load.
Like the experiments where the soil was applied to the wound,
the presence of nestmates was also effective in decreasing the
mortality of injured workers exposed to a known concentration of
P. aeruginosa (OD = 0.05). While the mortality of infected ants kept in
isolation was 93%, mortality of infected ants that had been returned
to their nestmates was only 8% (least square means: DF = 142;
Z=−2.94; P= 0.01; Fig. 2a, Supplementary Fig. 2c, and Supplemen-
tary Table 4). There were major differences in the increase in Pseu-
domonas load after injury between ants kept with or without their
nestmates (Fig. 2b & Supplementary Table 5). The bacterial load of
infected ants kept with their nestmates did not increase significantly
from 2 and 11 h after injury (least square means: DF = 18; t=−0.037,
P=1;Fig. 2b). By contrast, there was a 100-fold increase in bacterial
load for infected ants kept in isolation (least square means: DF = 18;
t=−4.832, P< 0.001; Fig. 2b).
Wound care behavior
To study the proximate mechanisms reducing mortality of ants
infected with P. aeruginosa when they are returned to their nestmates,
we introduced injured ants (with sterile and infected wounds) to their
nestmates and filmed them for 24 h. We observed that workers treated
the injury of infected ants by depositing secretions produced by the
metapleural gland (MG), which is located at the back of the thorax
(Fig. 3a). The MG secretions, which have antimicrobial properties13–17,
were applied in 10.5% of the wound care interactions (43 out of 411).
Before applying MG secretions, the nursing ant always groomed the
wound first (i.e., “licking”the wound with their mouthparts). Nursing
ants then collected the secretions either from their own MGs (Sup-
plementary Movie1), by reaching backto the opening of the glandwith
their front legs to collect the secretion and then licking their front legs
to accumulate it in theirmouth, as described in other species15, or from
the MG of the injured ant itself, by licking directly into the gland’s
opening (Supplementary Movie 2). Wound care with MG secretions
lasted significantly longer (85 ± 53 s) than wound care without MG
secretions (53 ± 36 s; t-test: DF = 47.92; t=−3.09; P=0.003; Supple-
mentary Fig. 4).
Remarkably, workers were able to discriminate between infec-
ted and sterile ants.Wound care treatment was provided more often
to infected ants upon initial introduction to the nest and again 10-
and 11-hours post-introduction (hierarchical generalized additive
model: P< 0.05; Fig. 3b). Moreover, MG secretions were deposited
significantly more often on wounds of infected than sterile ants
between 10 and 12 h after infection (hierarchical generalized additive
model: P< 0.05; Fig. 3c). When treatment with MG secretions was
prevented (through plugging the MG opening of all ants in the sub-
colony), mortality of infected ants reached 100% within 36 h com-
pared to only 33% when workers had access to the MG secretions
(least square means: DF = 47; Z=2.99; P= 0.039; Fig. 4, Supplemen-
tary Fig. 5, and Supplementary Table 6). By contrast, the plugging of
the MG opening had no significant effect on the survival of sterile
ants (least square means: DF = 47; Z = 0.02; P= 1; Fig. 4and Fig. S5 and
Table S6).
CHC profiles as infection cues
Because cuticular hydrocarbons (CHCs) are known to be frequently
used as a source of information in ants18, we investigated whether the
CHC profile of infected ants changed over the course of the infection.
Immediately after injury, infected ants did not differ from sterile ants
in their CHC profile (ADONIS: R2= 0.013; F
(1,11)
=0.13; P= 0.96; Sup-
plementary Tables 7 and 8). This profile changed in both types of ants
during the two hours after injury (Sterile ants: ADONIS: R2=0.15;
F
(1,16)
=2.74; P= 0.03; Infected ants: ADONIS: R2=0.13; F
(1,17)
=2.42;
P= 0.04), converging towards a similar profile for both types of ants
(ADONIS: R2= 0.008; F
(1,22)
=0.17;P= 0.87). Thereafter, the CHC pro-
file of infected ants remained unchanged until 11 h after injury (ADO-
NIS: R2=0.063;F
(1,23)
= 1.48; P=0.13), whilethe CHC profile of sterile
ants changed significantly (ADONIS: R2= 0.14; F
(1,22)
=3.54; P=0.04),
thereby becoming significantly different from the CHC profile of
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infected ants (ADONIS: R2=0.19;F
(1,23)
=5.4; P= 0.007). Eleven hours
after injury, the CHC profile of sterile ants had converged again to the
profile at 0 h (ADONIS: R2=0.03; F
(1,17)
=0.53;P=0.45),whiletheCHC
profile of injured ants remained significantly different (ADONIS:
R2=0.19; F
(1,17)
=3.82;P=0.008).
Consistent with the idea that changes in CHC profiles could pro-
vide cues on the health status of ants9, the observed differences in the
CHC profiles mostly stemmed from differences in the relative abun-
dance of alkadienes (Supplementary Fig. 6 and Supplementary
Table 9), which are among the CHC compounds most relevant for
communication in social insects19. These changes in the CHC profile
are generally regulated by differentially expressed genes in the fat
bodies20. To identify the genes likely responsible for the observed CHC
changes between infected and sterile ants, we conducted tran-
scriptomic analyses of the fat bodies of the same individuals. A total of
18 genes related to CHC synthesis were differentially expressed
between infected and sterile ants 11 h after exposure to P. aeruginosa
(in addition, 17 genes out of 378 that were differentially expressed were
Fig. 1 | Lethal effects and diversity of soil pathogens. a Relative 16 S rRNA gene
copies (bacterial load ΔCq) for individuals whose wounds were exposed to a sterile
PBS solution (blue: Sterile), or soil pathogens diluted in PBS (red: Infected, OD = 0.1),
2 and 11 h after exposure (n=10perboxplot,seeSupplementary Table 1 for statis-
tical results). Significant differences (P< 0.05) are shown with different letters and
were calculated using a two-sided least square means with Holm-Bonferroni cor-
rection. bAbsolute 16 S rRNA gene copy numbers summarized at the genus-level for
the 18 amplicon sequence variants (ASV) that had at least 1% relative abundance in
five of the 40 analyzed ants across the experiment (n= 10 per boxplot). Significance
is shown for pairwise comparisons between sterile (blue) and infected ants (red):
***=P< 0.001; **=P<0.01;*=P< 0.05; n.s.= not significant (P> 0.05). Detailed
statistical results in Supplementary Table 2, significant differences were calculated
with a two-sided permutation t-test with Holm-Bonferroni correction. (c) Kaplan –
Meier cumulative survival rates of workers in isolation (dotted line) or inside the nest
(solid line) whose wounds were exposed to a sterile PBS solution (blue: sterile), or
soil pathogens diluted in PBS (red: infected, OD = 0.1). Significant differences
(P< 0.05) are indicated with different letters. Detailed statistical results in Supple-
mentary Fig. 2a and Supplementary Table 3, significant differences were calculated
using a two-sided least square means with Holm-Bonferroni correction. Boxplots
show median (horizontal line), interquartile range (box), distance from upper and
lower quartiles times 1.5 inter-quartile range (whiskers), outliers (>1.5x upper or
lower quartile). Source data are provided as a Source Data file.
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immune genes; Fig. 5a and Supplementary Tables 10 and 11), while only
two genes related to CHC synthesis were differentially expressed two
hours after infection (in addition to 7 immune genes out of 164; Fig. 5b
and Supplementary Tables 10 and 11).
Antimicrobial content and efficacy of the Metapleural gland
To quantify the capabilities of MG secretions to inhibit bacterial
growth, we conducted antimicrobial assays. The growth of P. aerugi-
nosa was reduced by >25% when MG secretions were included in a
lysogeny broth (LB) solution compared to a control LB solution with-
out the MG secretions (Mann-Whitney U test: W=54,
P< 0.001, Fig. 3d).
Since P. aeruginosa has repeatedly developed antimicrobial
resistance21 and because most antimicrobial compounds found in
animal saliva are unable to inhibit the growth of P. aeruginosa22,we
conducted proteomic and chemical analyses of the MG secretions.The
proteomic analysis revealed 41 proteins (Supplementary Fig. 7 and
Supplementary Table 12), 15 of which showed molecular similarity to
toxins, which often have antimicrobial properties23.Fiveproteinshad
orthologs known to have antimicrobial activity (e.g., lysozyme,
hemocytes, 2 MRJP1-like proteins) and three with melanization, a
process implicated in wound healing in insects24,25. There was no clear
function for nine proteins including the most abundant protein
(13 ± 16% of the MG’s endogenous protein content), for which no
ortholog could be found. The evolutionarily young gene coding this
protein could be a promising candidate for antimicrobial research,
with research into next-generation antibiotics often relying on anti-
microbial peptides26. The gas-chromatography/mass-spectrometry
(GC-MS) analyses of the MG further revealed 112 organic compounds
(23 of which could not be identified, Supplementary Fig. 8 and Sup-
plementary Table 13). Six of the identified compounds had antibiotic-
and/or fungicide-like structures and 35 were alkaloids. While we could
not identify the exact structure of the alkaloids, many of them are
known to have antimicrobial properties27. There were also 14 car-
boxylic acids, making up 52% of the secretions content (Supplemen-
tary Fig. 8 and Supplementary Table 13), probably leading to a lower
pH detrimental to bacterial survival and growth13.
The number of chemical compounds identified in the MG’s
secretions of M. analis (112) is far greater than in other ant species,
where the number of compounds ranges between 1 and 35 and mostly
consists of carboxylic acids rather than alkaloids or antibiotic-like
compounds13. In other ant species, the compounds in MG secretions
are generally believed to be effective against early developmental
stages of pathogens, like during sterilization of the nest or as a
response to recent fungal exposure13–15. While we also observed a
prophylactic use of MG secretions directly after injury, the most
intense use of the MG was at the height of the infection (10–12 h,
Fig. 3c), a period where, without care,infected antsstart to die (Fig. 2a).
This suggests that, in addition to prophylactic substances, the MG
secretions also contain antimicrobial compounds capable of combat-
ting festering infections.
During wound care, the use of the MG secretions probably fulfils a
similar role as the mammals’antiseptic saliva. This analogous role
likely led to the convergent evolution of functionally similar anti-
microbial and wound healing proteins28. However, wound care treat-
ments are indiscriminate in mammals as they have never been
observed to depend on the infected state of the wound.
This study reveals an effective behavioral adaptation to identify
and treat festering infections of open wounds in a social insect. The
prophylactic and therapeutic use of antimicrobial secretions to
counteract infection in M. analis (Fig. 3c) mirrors modern medical
procedures used for treating dirty wounds29. Remarkably, the primary
pathogen in ant’s wounds, Pseudomonas aeruginosa, is also a leading
cause of infection in combat wounds, whereinfections can account for
45% of casualties in humans30.InM. analis the targeted treatment with
antimicrobial compounds was extremely effective in preventing lethal
Fig. 2 | Survival probability and pathogen load of sterile and infected ants.
aKaplan–Meier cumulative survival rates of workers in isolation or inside the nest
whose wounds were exposed to P. aeruginosa diluted in PBS (Infected, OD = 0.05)
or a sterilePBS solution (Sterile). Detailed statistical results in Supplementary
Fig. 2c and Supplementary Table 3. brelative bacterial load (ΔCq) of Pseudomonas
at two different timepoints (2 h and 11 h) forants in isolationor inside the nest with
wounds treated the same way as in Fig. 2a (Infected or Sterile). n= 6 per boxplot,
significant differences (P<0.05) are shown with different letters (Supplementary
Table 4). Boxplots show median (horizontal line), interquartile range (box), dis-
tance from upper and lower quartiles times 1.5 inter-quartile range (whiskers),
outliers(>1.5x upper or lowerquartile). Significant differencesin both panels were
calculated using a two-sided least square means with Holm-Bonferroni correction.
Colorsin both panels showin red: infected antsin isolation,in light blue: sterileants
in isolation, in dark blue: sterile ants inside the nest, in green: infected ants inside
the nest. Source data are provided as a Source Data file.
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bacterial infections by P. aeruginosa. This could potentially lead to
promising new medical compounds to cure infections in human
societies.
Methods
The research conducted in this study complies with all relevant
ethical regulations and was approved by the park management of
Office Ivoirien desParcs et Réserves (OIPR) inCôte d’Ivoire as part of
the bilateral research agreement between Germany (represented by
the University of Würzburg) and Côte d’Ivoire (represented by OIPR).
The ants collected for this study are part of the bilateral research
agreement under research permit number N°018 / MINEDD /
OIPR / DZ.
Experimental design
The study was conducted ina humid savannah woodland located in the
Comoé National Park, northern Côte d’Ivoire (Ivory Coast), at the
Comoé National Park Research Station (8°46’N, 3°47’W)31. Experi-
ments, observations, and sample collections in the Comoé National
Park were carried out from April to June 2018, February, April to June
and September to October 2019, April 2020 and October 2022.
Megaponera analis is found throughout sub-Saharan Africa from 25°S
Fig. 3 | Use and efficacy of the metapleural gland (MG) secretions during
wound care.a Micro CT scan showing the location of the MG. Blue:secretory cells;
yellow: atrium. bProbability of receiving wound care over 24 h fitted with a hier-
archical generalized (binomial) additive model (HGAM); shaded bars indicate
periods during which the probability of receiving care was significantly (P<0.05)
higher for infected (red; n= 6) than sterile (blue; n= 6) individuals. The line
represents the predicted probability by the HGAM, with the colored shaded area
representing the 95% confidence interval. cProbability of receiving antimicrobial
wound care with metapleuralgland (MG) secretions (the same ants as in Fig. 3b),
modeled with identical HGAM specifications. dBacterial growth assay for P. aeru-
ginosa either in LB broth (positive control, n= 6) or LB broth with MG secretions
(Metapleural gland n= 9). Two-sided Mann-Whitney U test: W=54,P<0.001.
Boxplots show median (horizontal line), interquartile range (box), distance from
upper and lower quartiles times 1.5 inter-quartile range (whiskers), outliers (>1.5x
upper or lower quartile). Source data are provided as a Source Data file.
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to 12°N32 and known to show monophasic allometry within its worker
sizes33. We thus divided the workers into majors (head width > than
2.40 mm), minors (head width <1.99 mm), and intermediates (head
width 2.40 −1.99 mm). All experiments were carried out on the minor
caste, the individuals most frequently injured2.Allfield studies were
conducted in accordance with local legislation and permission by the
Office Ivoirien des Parcs et Réserves (OIPR).
Laboratory colonies
Eleven colonies, including queen and brood (colony size 1083 ± 258
ants), were excavated and placed in artificial nests in the field stations
laboratory. PVC nests (30x20x10 cm) were connected to a 1x1m
feeding arena. The ground surface was covered with soil from the
surrounding area (up to a height of 2 cm). Colonies were fed by placing
in the feeding arena Macrotermesbellicosus termitescollected fromthe
surrounding area. These termites were found by scouts and triggered
raiding behavior. The laboratory windows were kept open to maintain
a natural humidity, temperature, and day-night cycle (light regime).
Survival of injured ants
To quantify the lethality of various types of pathogens, workers were
injured by a sterile cut in the middle of the femur on the hind leg and
had the fresh wound submerged for 2s in a 10μL phosphate-buffered
saline (PBS) solution with a known pathogen concentration (~106
bacteria of either the mix of soil pathogens or isolated pathogen).
Afterwards the injured ants were placed inside a cylindrical glass
container with a diameter of 3 cm and a height of 5 cm. Before the
experiments, the glass containers were filled with 1 cm of surface soil
andplacedfor3hat220°Cinanoventogetherwiththeforcepsand
scissors for sterilization. Nest-like humidity was created by moistening
the soil with 1 mL of sterilized water (boiled for 10min) and covered
with aluminum foil. The isolation experimentswere conducted at 24 °C
in a sterile room. To test for mortality, the injured ant was checked
upon once per hour for the next 36 h, if no reaction was observed even
after shaking the container the ant was classified as dead. To ensure
replicability of the survival experiments a negative (sterile PBS solu-
tion) and positive control (using a sterile PBS solution with 0.1 optical
density (OD) of Pseudomonas aeruginosa (PSE)) were always included
during each survival experiment. The sample sizes were PBS: n=104;
PSE 0.1: n=61;PSE0.05:n=15 (Fig.2a and Supplementary Fig. 3). For
all sample sizes and statistics see Supplementary Fig. 2.
To test if wound care by nestmates reduces the mortality of
infected ants, we first marked 10 ants per colony during a raid. 24 h
later, all marked ants were injured in the same way as in the isolation
trial. Afterwards, the wound of the injured ant was either exposed to
a sterile PBS solution (sterile), a sterile PBS solution containing 0.05
OD of P. aeruginosa or a sterile PBS solution containing 0.1 OD of
surface soil pathogens (grown on agar plates). Isolation trials with
the same treatments were always conducted in parallel. Sample size
of in nest survival experiments: PBS: n= 12; PSE 0.05: n= 12; Soil
n= 18 (Figs. 1and 2a). For all sample sizes and statistics see Supple-
mentary Fig. 2.
To test the importance of the Metapleural gland (MG) secretions
we divided three colonies (~1000 ants) into two equally sized sub-
colonies including brood. The queens were left with a small retinue of
20 workers in a separate container. For each colony we plugged the
MG opening of all workers ofone of the sub-colonies with acrylic color,
while forthe other sub-colony acrylic color was placed on the thoraxof
the workers. Afterwards the experimental procedure was identical to
the wound care experiment described above. Eight individuals were
removed from each sub-colony, marked, and wounded and the wound
exposed to either a sterile PBS solution (n=4 per sub-colony) or a
sterile PBS solution containing 0.05 OD of P. aeruginosa (n=4persub-
colony). Samplesizes were thus in total: Sterile: n= 12; Infected: n=12
for both treatments (with and without plugged MG opening; Fig. 4). In
addition, to quantify any effects of the plugging of the MG opening on
individuals, a parallel experiment with n= 12 Sterile and n=12Infected
ants with or without plugged MG opening was run in isolation (Sup-
plementary fig. 5 and Supplementary Table 6).
The pathogen concentration was measured by optical density
(OD) using a portable Ultrospec 10 cell density meter (Biochrom) with
sterile PBS as solvent. For the soil pathogens, we collected surface soil
in the surrounding area of the nest and grew it over 36 h on agar plates
(Supplementary Fig. 1c). For P. aeruginosa we created cultures of the
isolated strains (Supplementary Fig. 1d) in the field lab from frozen
samples kept in Tryptic Soy Broth (TSB) medium with 25% glycerol
(stored and transported at −23 °C). After replating the bacterial culture
once on a fresh plate, we waited 16 h before applying the pathogen on
fresh wounds. For all experiments, we used trypticase soy agar (TSA)
plates to culture the bacteria.
Treatment of wounds by nestmates
To quantify the wound care behaviors inside the nest, we filmed the
ants using a Panasonic HC-X1000 and analyzed the videos using VLC
media player v.3.0.16 Vetinari (intel 64 bit). The wound of the injured
ant was either exposed to a sterile PBS solution (Sterile) or a sterile PBS
solution containing 0.05 OD of P. aeruginosa (Infected).
All manipulated ants were placed in front of the nest entrance
directly after a raid and the nest was filmed for the subsequent 24 h.
Only one trial was conducted per colony with a total of four injured
ants per colony (two sterile and two infected), in a total of three
colonies (i.e., n= 6 infected ants and n= 6 sterile ants, Fig. 3b, c). The
observed wound care behaviors were classified into two categories:
(1) wound care: a nestmate cleans the open wound with its mouthparts;
(2) metapleural gland (MG) care: a nestmate collects MG secretions
either from its own gland (Supplementary Movie 1) or from the injured
ant (Supplementary Movie 2) in its mouth before caring for the wound.
a a
a
b
0
25
50
75
100
0 4 8 12162024283236
Time [h]
Survival probability [%]
Sterile
Infected
Plugged MG
Open MG
+
+
+
+
Fig. 4 | Effect of MG secretions on survival of sterile and infected ants.
Kaplan–Meier cumulative survival rates of workers inside sub-colonies with a
plugged metapleuralgland (MG) opening (dotted line) or with an unmanipulated
MG opening (solidline) whose wounds were exposed to a sterile PBS solution (blue,
sterile n=12)orP. aeruginosa diluted in PBS (OD = 0.05) (red, infected n=12).
Detailed statistical results in Supplementary Fig. 2d and Supplementary Table 6,
significant differences were calculated using a two-sided least square means with
Holm-Bonferroni correction. Additional data for ants in isolation can be found in
Supplementary Fig 5. Source data are provided as a Source Data file.
Article https://doi.org/10.1038/s41467-023-43885-w
Nature Communications | (2023) 14:8446 6
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These behaviors were quantified for the first 24h and summarized in
10 min intervals.
Sample collection protocol for chemical, genetic, and microbial
analyses
To quantify the cuticular hydrocarbon (CHC) profile (Supplementary
Fig. 6), differential gene expression (Fig. 5), and pathogen load in the
thoraxcontent (Figs.1aand2b), we usedthe same experimentaldesign
as used to quantify the survival of injured ants (see above). In total,
30 sterile and 30 infected ants (P. aeruginosa OD = 0.05) were pre-
pared, 12 sterile and 12 infected ants were kept in isolation, another
12 sterile and infected ants wereplaced inside the nest and 6 sterile and
6 infected ants were collected immediately after injury. Sterile or
infected ants were then collected from their enclosures at either 2 or
11 h after manipulation (n=6 per treatment). Only ants that did not die
until these time points were used for further analyses. The collected
ants were then first placed in hexane for 10min to extract the CHC
profile. The gaster wasthen cut off and placed in RNAlater for genetic
analyses and the thorax was placed in 100% ethanol for the microbial
analyses. The samples for genetic and microbial analyses were brought
to the University of Lausanne and the samples for chemical analyses to
the University of Würzburg. Another 10 sterile and 10 infected ants
kept in isolation were further observed for a total of 36 h to ensure that
the survival curves in this experiment resembled those in Fig. 2a.
Pathogen identification & isolation
To isolate potential pathogens, we collected surface soil in the sur-
rounding area of the nest and grew its water extract on Sarborough
Dextrose Agar (SDA) plates for 36 h at local temperature to get the ‘soil
pathogen mix’(Supplementary Fig. 1c). Most of the plate was at first
overgrown by black mycelia identified as Rhizopus microsporus.This
fungus is known to contain symbiotic bacteria Burkholderia sp. (spe-
cies not identified), as we confirmed by Sanger sequencing and bac-
terial community analysis (Fig. 1c). After several days, the culture
started to show several large colonies of slimy microorganisms that
were isolated and identified as Pseudomonas aeruginosa (Supplemen-
tary Fig. 1d). For infection assays (Supplementary Fig. 3), we isolated
Burkholderia and its fungal host Rhizopus from each other by repeated
passaging with antifungal nystatin (0.25 μl/ml in M9 agar, 30 °C) or
antibacterialciprofloxamin (0.02 mg/ml in SDA). No representatives of
Klebsiella were isolated.
The identification of all microorganisms was done by preparation
of a PCR-ready DNA from a piece of biomass as described in Lõoke
et al.34. PCR reactions were done with universal fungal pr imers ITS5 5’-T
Fig. 5 | Differentialgene expression betweensterile and infected ants 2 and 11 h
after injury. Infection triggers changes in the expression of hundreds of genes.
Volcano plot illustrates the fold up- and down-regulation of immune-related genes
(red triangles, Supplementary Table 11) and lipids and CHC-related genes (yellow
squares, Supplementary Table 10), 11 h (a)and2h(b) after infection. Positive
Log2FoldChange values correspond to genes up-regulated ininfected ants when
compared to sterile ants, whilenegative values are down-regulated in infected ants.
Significant differences werecalculated using a two-sided Wald test and corrected
for multiple testing using the Benjamini and Hochberg method (genes with sig-
nificant differential expression are marked in blue, i.e. adjusted P-value < 0.05).
Source data are provided as a Source Data file.
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CCTCCGCTTATTGATATGC-3′and ITS4 5′-GGAAGTAAAAGTCGTAAC
AAGG-3′35 (GoTaq polymerase, Tm = 55°C, elongation for 40s) and
commonly used universal bacterial primers 27F 5′-AGRGTTYGAT
YMTGGCTCAG-3′and 1492 R 5′-GGTTACCTTGTTACGACTT-3’(same
protocol but elongation for 1min 20 s). The presence of PCR ampli-
cons was verified on electrophoresis gel and the fragments were sent
for Sanger sequencing. The resulting sequences were then screened
against NCBI database with nucleotide BLAST36.
Microbiome analysis and bacterial load quantification
To quantify the composition of the bacterial community in workers,
we used the Powersoil DNA isolation kit (MO Bio) to extract DNA from
M. analis thoraxes kept in RNAlater. Samples were homogenized by
vortexing for 10 min, followed by twotimes 45 sec bead beating at6 m/
s using a FastPrep24TM 5 G homogenizer. Then we continued according
to the kit’s manual and eluted DNA in 100 µLofnuclease-freewater.
Bacterial loads were quantified with a QuantStudio5 qPCR
instrument (Applied Biosystems) using the reaction set-up described
in Kešnerová et al.37 and the thermal cycling conditions recomm
ended for SYBR® Select Master Mix. For quantifying total bacterial
loads, we used our designed primers #1047 5’-AGGATTAGAT
ACCCTRGTAGTC-3’and #1049 5’-CATSMTCCACCRCTTGTGC-3’(at
doubled 0.4 µM concentration). For specific targeting we used P.
aeruginosa primers #1209 5’-GTAGATATAGGAAGGAACACCAG-3’and
#1210 5’-GGTATCTAATCCTGTTTGCTCC-3’and for normalization to
the host’s housekeeping gene we used M. analis 28S rRNA gene pri-
mers #1207 5’-CTGCCCGGCGGTACTCG-3’and #1208 5’-A
CCGGGGACGGCGCTAG-3’. Serial dilutions (10x) showed that these
primers performed with an amplification efficiency (E) of 1.86
(R2= 0.99), 2.00 (R2= 0.99), and 1.87 (R2=0.99),respectively.
Total bacterial (Fig. 1a) and P. aeruginosa (Fig. 2b) 16 S rRNA gene
(target) copy numbers were expres sed relatively to M. analis 28 S rRNA
gene copy numbers (host) based on the following equation: ΔCq=
2^(Cq
host
-Cq
target
),whereCqwasthemeasured‘quantification cycle’
value. To calculate the total 16 S rRNA gene copy number in 1 μlofeach
DNA sample that was used for absolute bacterial abundancebased on
ASV counts (Fig. 1b) we used the equation n = E^(intercept –Cq)38,
where standard curve’sintercept =38.23
37.
16 S rRNA gene amplicon-sequencing
16 S rRNA gene amplicon sequencing data were obtained from 40
experimental samples, a mock sample (to verify consistency of the
MiSeq run compared to previous studies in our group), two blank
DNA extractions and a negative PCR control with only H
2
O. We fol-
lowed the Illumina 16 S metagenomic sequencing preparation guide
(https://support.illumina.com/documents/documentation/chemistry_
documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.
pdf) to amplify a nd sequence the V4 region of the 16 S rRNA gene.
Primers for the first PCR step were 515F-Nex (TCGTCGGCAGCGTCAG
ATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA) and 806R-Nex
(GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGG
TWTCTAAT). PCR amplifications were performed in a mix of 12.5 μLof
Invitrogen Platinum SuperFi DNA Polymerase Master Mix, 5 μLof
MilliQ water, 2.5 μL of each primer (5 μM), and 2.5 μL of template DNA.
PCR conditions were 98 °C for 30 s, 25 cycles of 98 °C for 10 s, 5 5 °C for
20 s, and 72 °C for 20 s, and a final extension step at 72 °C for 5 min.
Amplifications were confirmed by 2% agarose gel electrophoresis. The
PCR products were then purified with Clean NGS purification beads
(CleanNA) in a 1:0.8 ratio of PCR product to beads, and eluted in
27.5 μL of 10 mM Tris, pH 8.5.We then performed a second PCR step in
which unique dual-index combinations were appended to the ampli-
cons using the Nextera XT index kit (Illumina). Second-step PCRs were
performed in a 25 μL, using 2.5 μLofthePCRproducts,12.5μLof
Invitrogen Platinum SuperFi DNA Polymerase Master Mix, 5 μLof
MilliQ water, and 2.5 μLofeachoftheNexteraXTindexingprimers1
and 2. PCR conditions were 95 °C for 3min followed by eight cycles of
30 s at 95 °C,30 s at 55°C, and 30s at 72°C, and a final extension step
at 72 °C for 5 min. The libraries were again purified using Clean NGS
purification beads in a 1:1.12 ratio of PCR product to beads, and eluted
in 27.5 μL of 10 mM Tris, pH 8.5. The amplicon concentrations,
including the negative PCR control, mock, and blanks, were quantified
by PicoGreen and pooled in equimolar concentrations (except for the
water and blank samples that were kept at lower concentrations). We
verified that the final pool was of the right size using a Fragment
Analyzer (Advanced Analytical). MiSeq (Illumina) sequencing was then
performed at the Genomic Technology Facility of the University of
Lausanne, producing (2 × 250bp) reads. We obtained a total of 979’126
paired-end reads across the 40 experimental samples.
Analyses of 16 rRNA gene amplicon-sequencing data
Raw sequencing data (deposited at the Sequence Read Archive (SRA)
under PRJNA826317) were analyzed with the Divisive Amplicon
Denoising Algorithm 2 (DADA2) package v.1.20.0 in R. All functions
were run using the recommended parameters (https://benjjneb.
github.io/dada2/tutorial.html)exceptforthefiltering step in which
we truncated the forward and reverse reads after 232 and 231bp,
respectively. At the learnErrors step, we then set randomize=TRUE and
nbases=3e8. Amplicon-sequence variants (ASVs) were classified with
the SILVA database (version 138). Unclassified ASVs and any ASV
classified as chloroplast, mitochondria or Eukaryota were removed
with the “phyloseq”package version 1.36.0, using the “subset taxa”
function. We then used the “prevalence”method in the R package
“decontam”v.1.12.0 to identify and remove contaminants introduced
during laboratory procedures, using the negative PCR control and the
blank samples as reference. This procedure filtered out three con-
taminant ASVs. Four additional ASVs were removed as they clearly
represented (low abundance) contaminants due to index swapping
from samples of a different project that were sequenced in the same
sequencing run. We then calculated absolute bacterial abundances of
each ASV by multiplying the proportion of each ASV by the total 16S
rRNA gene copy number of each sample as measured by qPCR. To
assess differences in community structure between treatments we ran
ADONIS tests after calculating Bray-Curtis dissimilarities with the
absolute ASV abundance matrix and plotted ordinations based on
these Bray-Curtis dissimilarities (Supplementary Fig. 1a).
To test for differences in absolute abundance of individual bac-
terialgenera between sterile and infected ants (Supplementary Fig. 1b),
we used a permutation approach (permutation t-test) as done in Keš-
nerová et al.39. To do this, we selected the ASVs that had at least 1%
relative abundance across five samples (18 ASVs belonging to 11 gen-
era). We then calculated copy numbers at the genus-level for each of
the 40 samples. At each time-point (2h and 11h), we randomized the
values of the calculated copy numbers for each genus 10,000 times
and computed the tvalues for the tested effect for each randomized
dataset. The Pvalues corresponding to the effects were calculated as
the proportion of 10,000 tvalues that were equal or higher than the
observed one.
Chemical analysis of cuticular hydrocarbons
To quantify differences in CHC profiles between infected and sterile
ant workers, cuticular hydrocarbon extracts were evaporated to a
volume of approximately 100 μLand1μL was analyzed by using a
6890 gas chromatograph (GC) coupled to a 5975 mass selective
detector (MS) by Agilent Tec hnologies (Waldbronn, Germany). The GC
was equipped with a DB-5 capillary column (0.25mm ID × 30m; film
thickness 0.25 μm, J & W Scientific, Folsom, Ca, USA). Helium was used
as a carrier gas with a constant flow of 1 mL/min. A temperature pro-
gram from 60 °C to 300°C with 5°C/min and finally 10minat300°C
was employed. Mass spectra were recorded in the EI mode with an
ionization voltage of 70eV and a source temperature of 230°C. The
Article https://doi.org/10.1038/s41467-023-43885-w
Nature Communications | (2023) 14:8446 8
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software ChemStation v. F.01.03.2357 (Agilent Technologies, Wald-
bronn, Germany) for windows was used for data acquisition. Identifi-
cation of the components was accomplished by comparison of library
data (NIST 17) with mass spectral data of commercially purchased
standards and diagnostic ions.
To compare the relative abundances of the different compound
groups (Supplementary Fig. 6), all compounds were identified (Sup-
plementary Table 8) and grouped either into Alkanes, Alkenes, Alka-
dienes or Methyl-branched alkanes for each individual.
Antimicrobial assay
To assess the antimicrobial efficacy of MG extracts (Fig. 3d), we
quantified the increase over timeof the OD in a 96-well plate box, with
the outer row filled with 70 μL of PBS. In total we did 3 replicates per
sample with a total of 70 μL per sample. Negative control: 70 μL Luria-
Bertani (LB) broth (n= 6). Positive control: 68 μL LB broth +
2μLP. aeruginosa (n= 6). MG sample: 66 μLLBbroth+2μLP. aeru-
ginosa +2μLMGsample(n=9).
For the preparation of the pathogen sample, we plated P. aeru-
ginosa from a frozen stock on TSA plates. After 24h the bacterial
culture was replated. After 12h aliquots were created using the new
bacterial culture in a flask containing 15 mL of freshly sterilized LB
broth for an initial OD reading. Afterwards the flask was placed in an
incubator shaker (Amerex Steadyshake 757) at 180 rpm, 30 °C for the
bacteria to grow. Once the OD readings were between 02–0.5 OD (the
exponential growth phase) the flask was put on ice until the experi-
ments started.
For the preparation of theMG sample, we pooled 10 MGs in 1.5 mL
Eppendorf-Cups. We then froze the samples in liquid nitrogen and
crushed the sample material using sterile pellets. We then added 50 μL
of PBS-Buffer, vortexed shortly before centrifuging the sample for
5 min at 3000 x g at 4 °C. We then extracted 30μL of the supernatant
into a new cup and repeated the centrifugation process. Afterwards
20 μL of the supernatant was placed again in a new cup and stored at
−20 °C. Preparation of the wells was conducted on ice at 4 °C to pre-
vent bacterial growth and degradation of the MG samples. The anti-
microbial assays were done in a microplate reader (Synergy H1 BioTek)
at 30°C with a 600nm wavelength for 8 h with a double orbital
shaking step after each OD reading cycle (every 10 min) at the Uni-
versity of Lausanne.
To calculate the intrinsic growth rate of the microbial population
(r) in Fig. 3d we used the package growthcurver (v. 0.3.1) with the
statistical software R v4.1.0. “r”represents the growth rate that would
occur if there were no restrictions imposed on total population size.
Sample collection for metapleural gland extracts
Due to the difficulty of collecting adequate amounts of metapleural
gland secretions from the gland’s atrium (it is a very sticky substance
that adheres to the cuticle), we decided to remove the atrium of the
gland together with the secretory cells entirely, using a microscalpel
and microscissors. To avoid using a solvent we used a Thermodesorber
unit coupled to a GC-MS (TD-GC-MS). For this we transported frozen
ants to the University of Würzburg and did the extractions directly in
the laboratory next to the TD-GC-MS. One metapleural gland from
each of six worker was pooled per sample. As a control we further
collected pieces of cuticle of similar size from the side of the thorax to
identify any potential contaminations which might have occurred
during dissections. In total 3 samples (of 6 individuals each) were
analyzed (Supplementary Fig. 8, Supplementary Table 13).
Chemical analysis of the metapleural gland
The MG samples were placed in a glass-wool-packed thermodesorp-
tion tube and placed in the thermodesorber unit (TDU; TD100-xr,
Markes, Offenbach am Main, Germany). The thermodesorption tube
was heated up to 260°C for 10min. The desorbed components were
transferred to the cold trap (5 °C) to focus the analytes using N
2
flow in
splitless mode. The cold trap was rapidly heated up to 310 °C at a rate
of 60 °C per minute, held for 5 min and connected to the GC-MS
(Agilent 7890B GC and 5977 MS, Agilent Technologies, Palo Alto, USA)
via a heated transfer line (300 °C). The GC was equipped with an HP-
5MS UI capillary column (0.25 mm ID × 30 m; film thickness 0.25 μm, J
&WScientific, Folsom, Ca, USA). Helium was the carrier gas using
1.2874 ml/min flow. The initial GC oven temperature was 40 °C for
1 min, then raised at a rate of 5 °C per min until reaching 300 °C, where
it was held for 3min. The transfer line temperature between GC and
MS was 300°C. The mass spectrometer was operated in electron
impact (EI) ionization mode, scanning m/z from 40 to 650, at 2.4 scans
per second. Chemical compounds were identified using the same
protocol as for the CHCs.
Proteomic analysis and sample preparation of the
metapleural gland
To characterize the proteins secreted by the metapleural gland, we
analyzed the proteome of the atrium of the gland, the metapleural
gland secretory cells and the hemolymph (Supplementary Fig. 7). To
be considered a metapleural gland protein that could mediate anti-
microbial activity, proteins had to be found in the gland’s atrium, and
must have a higher abundance in the gland’satriumthaninthe
hemolymph. The samples for proteomic analysis were collected in
April 2020 from workers of field colonies collected in the Comoé
National Park. Three types of samples were collected: Secretory: dis-
sected secretory cells without the atrium, (n= 6 samples, each pooled
from five dissections); Hemolymph: hemolymph was collected from
the thorax by a glass microcapillary through a small wound
(n= 6 samples, each pooled over five individuals, yielding 4–5μL);
Atrium: due to the difficulty of collecting pur e MG content we chose to
first widen the opening of the atrium and add 1μl of PBS before
extracting the content together with the PBS (n= 6 samples, each
pooled from five individuals). The samples were kept at −23 °C in Lo-
bind Eppendorf tubes with 5 μl of PBS together with a 1x Protease
inhibitor Cocktail (Sigmafast) during transportation. Once in Lau-
sanne, Switzerland, the samples were kept at −80 °C and processed
swiftly.
Proteins were digested according to a modified version ofthe iST
protocol40. Samples were resuspended in 20 μLofmodified iST buffer
(1% sodium deoxycholate, 10 mM DTT, 100 mM Tris pH 8.6) and
heated at 95 °C for 5 min. They were then diluted with 24μLof4mM
MgCl
2
and 1:100 of benzonase in H
2
O and incubated 15min at ambient
temperature. 14 μL of 160 mM chloroacetamide (in 10 mM Tris pH 8.6)
were then added and cysteines were alkylated for 45 min at 25 °C in the
dark. After addition of 0.5 M EDTA (3 mM final concentration), samples
were digested with 0.1 μg of trypsin/Lys-C mix (Promega) at 37 °C for
1 h, followed by a second enzyme addition (0.1 μg trypsin/LysC) and 1 h
incubation. Two volumes of isopropanol + 1% trifluoroacetic acid (TFA)
were added to one volume of sample and loaded onto an equilibrated
OASIS MCX uElution plate (Waters) prefilled with SCX0 buffer (20%
MeCN, 0.5% formic acid) and centrifuged. The columns were washed
three times with 200 μL isopropanol + 1% TFA and once with 200 μL
HPLC solvent A (2% MeCN, 0.1% formic acid). The peptide mixturewas
then sequentially eluted with 150μL SCX125 buffer (20% MeCN, 0.5%
formic acid, 125mM ammonium acetate), 150 μL SCX500 buffer (20%
MeCN, 0.5% formicacid, 500mM ammonium acetate), and lastly with
150 μL basic elution buffer (80% MeCN, 19% water, 1% NH3).
Tryptic peptides fractions were dried and resuspended in 0.05%
trifluoroacetic acid, 2% (v/v) acetonitrile, for mass spectrometry ana-
lyses. Tryptic peptide mixtures were injected on an Ultimate RSLC
3000 nanoHPLC system (Dionex, Sunnyvale, CA, USA) interfaced to an
Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific, Bre-
men, Germany). Peptides were loaded onto a trapping microcolumn
Acclaim PepMap100 C18 (20 mm × 100 μmID,5μm, 100 Å, Thermo
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Scientific) before separation on a reversed-phase custom-packed
nanocolumn (75 μmID×40cm,1.8μm particles, Reprosil Pur, Dr.
Maisch). A flow rate of 0.25 μL/min was used with a gradient from 4 to
76% acetonitrile in 0.1% formic acid (total time: 65min). Full survey
scans were performed at a 120’000resolution,andatop-speedpre-
cursor selection strategy was applied to maximize acquisition of
peptide tandem MS spectra with a maximum cycle time of 0.6 s. HCD
fragmentation mode was used at a normalized collision energy of 32%,
with a precursor isolation window of 1.6 m/z, and MS/MS spectra were
acquired in the ion trap. Peptides selected for MS/MS were excluded
from further fragmentation during 60 s.
Tandem MS data were processed by the MaxQuant software
(version 1.6.14.0)41 using the Andromeda search engine42 matching to a
custom-made protein database containing 19’618 sequences of M.
analis (July 2019 version, see section “Genome annotation”for details),
supplemented with sequences of common contaminants. Trypsin
(cleavage at K,R)was used as the enzymedefinition, allowing 2 missed
cleavages. Mass tolerance was 4.5 ppm on precursors (after recali-
bration) and 0.5 Daon MS/MS fragments. Onlypeptides witha minimal
length of 7 were considered for protein identifications. Carbamido-
methylation of cysteine was specified as a fixed modification.
N-terminal acetylation of protein and oxidation of methionine were
specified as variable modifications. All identifications were filtered at
1% FDR at both the peptide and protein levels with default MaxQuant
parameters. For protein quantitation the iBAQ values43 were used.
MaxQuant data were further processed with Perseus software (version
1.6.14.0)44 for filtering, removing contaminants and proteins identified
with only 1 peptide, log2-transformation, normalization of values, and
ortholog annotations.
To determine which proteins are secreted by the MG, we pro-
ceeded through a series of filters. Proteins not found in the atrium
samples were eliminated and those found only in the MG atrium were
considered hits. The log2 fold change between the atrium and the
hemolymph were determined for all remaining proteins. Only proteins
that were on average 1.5-fold more abundant in atrium samples than
hemolymph were retained as hits. Percent of total iBAQper sample was
assigned to each protein to indicate abundance. Visualization of
heatmaps was performed in Matlab 2020b (clustergram function). To
determine whether our selected proteins were toxin-like, we ran
clantox45 (http://www.clantox.cs.huji.ac.il/). Annotations (function,
orthology depth, and implications for wound healing) found in Sup-
plementary Table 12 were determined using protein BLAST with the
experimental clustered nr database46.
The mass spectrometry proteomics data are deposited at the
ProteomeXchange Consortium via the PRIDE partner repository with
the dataset identifier PXD033003.
Genome sequencing and assembly
ThegenomeofM. analis was sequenced and assembled by The Global
Ant Genomics Alliance (GAGA, antgenomics.dk)47.Toidentifyrepeats
in the genome assembly, we used the package RepeatModeler v2.0,
which combines three de-novo repeat finding programs (RECON,
RepeatScout and LtrHarvest/Ltr_retriever)48. RepeatModeler first
probes chunks of the genome assembly to find repeats, then clusters
and classifies identified repeats, producing a high-quality library of
consensus sequences of repeated sequence families. The consensus
sequences were used with RepeatMasker to softmask (in lower-case)
all repeats in the genome assembly.
Genome annotation
To annotate the M. analis genome assembly we used the ab-initio gene
predictors Augustus v3.3.349 and genemark50 within the BRAKER2
pipeline v2.1.451,52. Both tools were trained using RNA-seq reads (see
below) aligned to the softmasked version of the genome with STAR
v2.753 and protein evidence from NCBI refseq predictions for
Acromyrmex echinatior, Atta colombica, Camponotus floridanus, Dino-
ponera quadriceps, Linepithema humile, Monomorium pharaonis,
Ooceraea biroi, Pogonomyrmex barbatus, Solenopsis invicta, Tem-
nothorax curvispinosus, Vollenhovia emeryi and Wasmannia aur-
opunctata. To enable functional interpretation of experimental data,
we annotated gene products. First, we identified gene orthologs in
Drosophila melanogaster to leverage the excellent quality of the
functional annotation in this species. To identify the orthologs, we
applied a reciprocal best blast approach using Orthologr54 between the
longest protein isoform for each gene from both species55. Addition-
ally, we identified orthologs with Apis mellifera and Camponotus flor-
idanus NCBI RefSeq gene annotations. Finally, we ran InterProScan
(version 5.30–69.0 and panther-data-14.1) on all proteins using default
settings. To assign functional information to M. analis annotated
genes, we primarily used the gene ontology of D. melanogaster
orthologs, but also results from sequence homology search on Inter-
proscan and the Uniprot database.
To study the effect of infections on gene expression (Fig. 5), we
conducted a transcriptomic analysis. We dissected the gaster from the
same infected (soil OD = 0.1, n= 20) and sterile ants (n=20), whose
thorax were used for the microbiome analyses in Fig. 1a, b. Samples
were collected 2 and 11 h after manipulation (n= 10 per timepoint) in
the Comoé field research station and had the sting together with the
venom reservoir removed. The gaster was dissected without solvent
and stored in a RNAlater stabilisation solution at −23 °C (ThermoFisher
Scientific) for later analyses at the University of Lausanne. Afterwards,
the samples were homogenized with ceramic beads in 1ml of Trizol
reagent. Homogenized samples were incubated for 5 min at room
temperature (RT) in Trizol, before adding Chloroform (200 μL). Sam-
ples were incubated for 5 min at RT then centrifuged (25 s at
12,000 rpm and 4 °C) and the upper aqueous layer (~500 μL) trans-
ferred to a new tube. We added Isopropanol (650 μL) and Glycogen
blue (1 μL; RNAse-free, Invitrogen, 15 mg/mL, #AM9516) then vortexed
and incubated overnight at −20 °C. To purify the RNA, we used a EtoH
precipitation method: samples were centrifuged (30s at full speed at
4 °C), the supernatant was discarded and EtOH (1mL at 80%) added.
We repeated this step a second time with EtOH (1mL at 70%). Finally,
the supernatant was removed and the pellet, after a brief air dry
(15–20 s) at RT, was resuspended in nuclease-free water. Libraries were
prepared with the KAPA Stranded mRNASeq Library Preparation Kit
(#KK8421) according to the manufacturer’s protocol. Paired end
sequencing was performed on an Illumina Hiseq4000 sequencer a t the
Genomic Technology Facility of the University of Lausanne. We
obtained ~15–25 million PE reads per individual. Sequence reads have
been deposited in NCBI Sequence Read Archive (SRA) under the
accession number PRJNA823913.
We mapped RNA-seq reads on the softmask genome assembly
using STAR v2.7 with the 2-pass mapping option and using the gene
annotations to properly find exonic junctions53. Then, we used the
program featureCounts from the Subread package to count the
number ofreads mapped on each exon for each gene56.Thenusingthe
count’sfiles, we performed differential gene expression analyses using
DESeq257 of sterile and infected workers collected at 2 and 11h after
treatment. Pvalues of differential expression analyses were corrected
for multiple testing with a false discover rate (FDR) of 5%.
X-Ray Micro-CT imaging
To examine the internal morphology of the MG (Fig. 3a), we scanned
andvisualizedanintermediate-sizedworker(CASENT0744096),
although multiple other workers of different sizes were scanned and
visually inspected to confirm the morphology of the exemplar speci-
men was representative. The specimens were fixed in 90% ethanol,
stained in 2 M iodine solution for a minimum of 7 days, then washed
and sealed in a pipette tip with 99% ethanol for scanning. The scans
were performed with the ZEISS Xradia 510 Versa 3D X-ray microscope
Article https://doi.org/10.1038/s41467-023-43885-w
Nature Communications | (2023) 14:8446 10
Content courtesy of Springer Nature, terms of use apply. Rights reserved

at the Okinawa Institute of Science and Technology Graduate Uni-
versity, Japan. We scanned the thorax (mesosoma) and whole body to
visualize both the structure of the gland, and its location in the whole
ant. The scan parameters for the thorax scan were: Voxel size,
4.404 μm; Exposure time, 6 s; Voltage, 40 kV; Power, 3 W; Source dis-
tance, 23.147 mm; Objective, 4x; Projections, 1601. For the full body
scan, we used: Voxel size, 12.5 μm; Exposure time, 1 s; Voltage, 40 kV;
Power, 3 W; Source distance, 25.03 mm; Objective, 0.4x; Projections,
1601. The 3D reconstruction was performed using the ZEISS Scout-and-
Scan Control System Reconstructor software (ZEISS Microscopy, Jena,
Germany). The MG structures were manually segmented using Amira
(version 2019.2; Thermo Fisher Scientific, Berlin, Germany) and
visualized using VGStudio3.4 (Volume Graphics GmbH, Heidelberg,
Germany). The gland atrium was rendered using isosurface and a
clipping plane was created to visualize the cross-section. All other
materials were visualized using Phong volume rendering.
Statistical analysis
For statistical analyses and graphical illustration, we used the statistical
software R v4.1.058 with the user interface RStudio v1.4.1717 and the R
package ggplot2 v3.3.559. To select the appropriate statistical tests, we
tested for deviations from the normal distribution with the
Shapiro–Wilks test (P> 0.05). A Bartlett test was used to verify homo-
scedasticity (P> 0.05). All our models included colony as a random
factor and an overall likelihood ratio test against an intercept only
model. In case of multiple testing, a Holm-Bonferroni correction was
performed with the adjusted P-values given throughout the text. To test
for significant differences in the survival curves, we conducted mixed
effect cox proportional hazards regression models (Supplementary
Fig. 2) using the R package survminer (v0.4.9) followed by post-hoc
analyses using least square means with the R package lsmeans (v.2.30;
Supplementary Tables 3, 4 and 6). The survival curves were illustrated
using Kaplan-Meier cumulative survival curves (Figs. 1c, 2a, and 4,and
Supplementary Figs. 3 and 5). For the bacterial load (ΔCq) within the
thorax content between treated and untreated infected individuals
(Figs. 1aand2b) we conducted linear mixed effect models with a least
square means (lsmeans) post-hoc analysis. For the bacterial growth
inhibition (Fig. 3d) we conducted a Mann–Whitney Utest. Differences in
the CHC-profile composition were calculated using a permutational
multivariate analysis of variance (ADONIS) on a Bray-Curtis dissimilarity
matrix using the package vegan (v.2.5–7; Supplementary Table 7). For
the different durations of MG care between sterile and infected indivi-
duals (Supplementary Fig. 4), we conducted a linear mixed effect model
with individual as random factor followed by a Satterthwaite’st-test.For
differences between chemical compound groups of the CHC profiles
(Supplementary Fig. 6), we conducted an analysis of variance (AOV)
followed by a Tukey Honest Significant differences test (Supplementary
Table 9). For behavioral differences in wound care between sterile and
infected individuals (Fig. 3b, c), we modeled wound care as a binary
event using binomial generalized additive models with posthoc con-
trasts to identify intervals of time during which the probability of
receiving wound care differed between sterile and infected individuals.
See Supplementary Information for a reproducible modeling workflow.
Ethics and inclusion statement
Our study brings together authors from a number of different coun-
tries but failed to include scientists based in the country where the
study was carried out. We recognize that more could have been done
to engage local scientists with our research as our project developed,
and to embed our research within the national context and research
priorities. Whenever possible, our research was discussed with local
stakeholders to seek feedback on the questions to be tackled and the
approach to be considered. Whenever relevant, literature publishedby
scientists from the region was also cited; efforts were made to consider
relevant work published in the local language.
Reporting summary
Further information on research design is available in the Nature
Portfolio Reporting Summary linked to this article.
Data availability
The raw amplicon-sequence data generated in this study have been
deposited at the Sequence Read Archive (SRA) under accession code
PRJNA826317. The sequence reads data generated in this study have
been deposited in NCBI Sequence Read Archive (SRA) under accession
code PRJNA823913. The proteomics data generated in this study have
been deposited at the ProteomeXchange Consortium via the PRIDE
partner repository under accession code PXD033003. The CHC and
MG data generated via GC-MS in this study have been deposited at the
Dryad repository under the https://doi.org/10.5061/dryad.hqbzkh1j6
[https://datadryad.org/stash/share/gFLLhPMhmWW8HJ_JaKyTqg9Eq3
QXqGS_EgxYEQvKKe8]60. The Genome Assembly data are available
under restricted access due to it being part of another Publication
within the GAGA Project47, access can be obtained by contacting the
corresponding Author of the GAGA Project. Source data are provided
as a Source Data file. Source data are provided with this paper.
Code availability
The R-code used in this study are provided in Supplementary data 1
and 2.
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Acknowledgements
We thank the Comoé National Park Research Station for the use of their
facilities for the field and laboratory research and the park management
of Office Ivoirien des Parcs et Réserves for facilitating field research in
the park. We thank GAGA for providing the assembled Megaponera
analis genome. We thank Barbara Milutinovic and the Cremer lab at IST
Austria for teaching us the methodology for the antimicrobial assay.
Article https://doi.org/10.1038/s41467-023-43885-w
Nature Communications | (2023) 14:8446 12
Content courtesy of Springer Nature, terms of use apply. Rights reserved

We thank David Kouassi and Abou Ouattara for their help with nest
excavation and colony maintenance in the field. We thank Christine La
Mendola for her help with sample preparation for genetic analyses and
Camille Lavoix for proof reading of the manuscript. This study was
supported by the Swiss NSF grant 310030_156732 and the ERC
Advanced Grant resiliANT (no: 741491) to LaK. ACL was supported by
Swiss NSF grant PR00P3_179776. ETF was supported by the DFG Emmy
Noether Programme (no: 511474012).
Author contributions
Conceptualization: E.T.F., La.K. Methodology: E.T.F., Lu.K., J.L., Q.H.,
A.C.L., A.D., F.A., E.P.E., P.W., T.S., and D.B.S. Investigation: E.T.F., Lu.K.,
J.L., Q.H., A.C.L., E.P.E., and T.S. Visualization: E.T.F., Q.H., F.A., E.P.E.,
A.C.L., J.L., and D.B.S. Funding acquisition: La.K. and E.T.F. Project
administration: E.T.F., P.E., and La.K. Supervision: E.T.F. and La.K. Writing
–original draft: E.T.F. and La.K. Writing –review & editing: E.T.F., Lu.K.,
J.L., Q.H., A.C.L., A.D., F.A., E.P.E., P.W., P.E., T.S., D.B.S., and La.K.
Funding
Open Access funding enabled and organized by Projekt DEAL.
Competing interests
The authors declare no competing interests.
Additional information
Supplementary information The online version contains
supplementary material available at
https://doi.org/10.1038/s41467-023-43885-w.
Correspondence and requests for materials should be addressed to
Erik. T. Frank or Laurent Keller.
Peer review information Nature Communications thanks Axel Touchard,
and the other, anonymous, reviewer(s) for their contribution to the peer
review of this work. A peer review file is available.
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