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The scent of symbiosis: gut bacteria affect social interactions in leafcutting ants

Animal gut microbiota affect host physiology and behaviour. In eusocial Hymenoptera, where colony-level integrity is preserved via a nestmate discrimination system based on cuticular hydrocarbon mixtures, microorganismal effects may influence social dynamics. Although nestmate recognition has undergone a thorough exploration during the last four decades, few studies have investigated the putative role of gut microbes. Here we integrate metagenomic, chemical and behavioural approaches to test whether gut microbes affect nestmate recognition in Acromyrmex echinatior leaf-cutting ants. Treating workers with a sterile diet or with antibiotics resulted in a substantial alteration of their gut microbial communities. In pairwise social interactions, untreated vs. antibiotic-treated nestmates behaved more aggressively than other nestmate and non-nestmate pairs, suggesting that the suppression of microbes indirectly alters chemical social cues and triggers aggressive behaviour. Chemical analyses on treated individuals revealed a decrease in the abundance of two metapleural gland antifungal compounds, and we confirmed the correspondence between aggression levels and chemical profile differences. Feeding microbiota-remodelled ants with conspecific faecal droplets partially restored the original bacterial communities. Furthermore, non-nestmates fed with faecal droplets from different colonies were unusually aggressive compared to pairs fed with faecal droplets from the same colony. This suggests that chemicals derived from microbial strains may shape nestmate recognition, opening novel questions about the role of microorganisms in the evolution of social behaviour.


Introduction
In the evolution of mutualistic relationships between metazoans and prokaryotes, 49 animals have co-opted the metabolic versatility of microbes to upgrade their 50 physiology, while microorganisms have found favourable environments in animal 51 bodies. As part of the physiology of their animal hosts, symbiotic microbes are 52 also involved in their behavioural processes (1-5). Many insightful discoveries 53 about the physiological and behavioural effects of symbiotic microorganisms 54 stem from the study of germ-free or germ-remodelled animals. Research 55 comparing germ-free mice to their untreated counterparts has revealed microbial 56 gut symbionts to affect anxiety-like behaviour (6-8) and social interactions (9). 57 Similarly, a flourishing corpus of Drosophila studies suggests that gut microbes 58 mediate a plethora of physiological/behavioural processes, including specific 59 compared the microbial communities of ant guts, heads and thoraxes before and 141 after the experimental diet treatment (details in the Supplementary Information, 142 Figure S2). 143

Aggression assays 145
We tested microbiota-remodelled and untreated ants against nestmates and 146 non-nestmates from the original colonies, in both experimental rounds (3-5 147 replicates/combination, total=260 tests; Figure 1, Data set S1). Assays consisted 148 of dyadic encounters in Petri dishes with clean filter paper on the bottom. For two 149 minutes after first contact, an observer (blind with respect to ant treatment and 150 original colony) used the software Etholog 2.25 (26) to quantify the frequency 151 and duration of biting, mandible opening, antennation and absence of contact 152 ( Figure S3). For statistical analyses, we excluded the mandible opening 153 behaviour (usually considered as aggressive) because pooling it with biting 154 produced similar results (Supplementary Information). In addition, because 155 keeping ants in the same Petri dishes for three weeks resulted in a significant 156 effect on aggression (Binomial GLMM with 'Petri dish' as fixed and 'colony' as nestmate, diet treatment and their interaction were included as fixed factors. The 165 colony origin of experimental individuals was included as random effect term. 166 The model used for Round 2 included the same response variable as Round 1 167 model, and 'faecal droplets' was included as an additional fixed factor (two 168 levels: from nestmates or non-nestmates). We tested the significance of fixed 169 effects using the car function 'Anova'. Where needed, we conducted post hoc 170 planned contrasts between groups of interest using the multcomp function 'glht', 171 correcting alpha values with false discovery rate (FDR control gene (37), always using zero as reference. We used linear mixed models 207 (LMM) with the ΔΔC T values as response variable, 'diet treatment' (i.e., 208 untreated, sugar-treated or tetracycline-treated) and 'experimental round' (i.e. 209 Round 1 or 2) as fixed variables, and 'colony' (A, B, C or D) as random variable. 210 Tukey post hoc tests were performed to evaluate significant differences between 211 groups. We used GLMs to pair aggression data with distances calculated using using the qPCR data. All correlations were performed in R using the lme4, 214 vegan, effects, Rmisc and ggplot2 packages (27,(38)(39)(40)(41). 215

16S rRNA MiSeq analyses 217
To investigate whether novel OTUs appeared with the dietary treatment, we 218 screened ant worker guts individually with 16S rRNA MiSeq sequencing. 219 Amplicons were generated using the 515F/806R primers targeting the 16S rDNA 220 'experimental round' as fixed variables, and 'colony' as a random variable. We 256 conducted linear hypotheses using the multcomp R package (44) function glht to 257 evaluate differences between diet treatments in the same Rounds, and between 258 the same diet treatments across Rounds. All p-values were corrected for false 259 discovery rate (FDR) given that we had conducted 73 separate tests per 260 contrast. We used a coinertia analysis (45) to check for correlations between six most abundant bacterial taxa (see details about these taxa below). In short, 263 we generated two independent data matrices (either using the individual profiles 264 or the pairwise differences for each trial), performed PCA analyses and paired 265 them using the coinertia analysis using a Monte-Carlo test with 10000 266 df=1, p=0.18; N=260 dyadic aggression assays). This result was mostly due to 283 the high biting frequency between tetracycline-treated ants and their untreated 284 former nestmates taken from fungus gardens, which was significantly higher 285 from all other nestmate trials (all p<0.05, Figure 2A, Table S2). For tetracycline-286 treated and sucrose-treated ants, aggression levels between non-nestmates 287 were not significantly different from those observed in nestmate trials 288 (tetracycline-treated: z=0.365, p=0.715, Figure 2A; sucrose-treated: z=1.284, 289 p=0.287). Contrarily, in the sucrose-vs. tetracycline-treated groups, non-290 nestmate trials showed higher aggression than nestmate trials (z=2.375,

Aggression tests (Round 2) 294
During Round 2, across sucrose-treated pairs, tetracycline-treated pairs and 295 sucrose-vs tetracycline treated pairs, aggression was low in nestmate 296 encounters fed with nestmate faecal droplets, higher in non-nestmate encounters 297 fed with nestmate faecal droplets and maximal in non-nestmate encounters fed 298 with non-nestmate faecal droplets (Figure 2b). In particular, the highest 299 aggression levels appeared in encounters between non-nestmate sucrose-300 treated ants fed with faecal droplets from different colonies (z=2.05, df=85, 301 p=0.041; Figure 2b, Table S2). Tetracycline-treated pairs showed instead low 302 aggression (Figure 2b), regardless of whether they were fed on the same or 303 different faecal droplets. Sucrose-treated vs tetracycline-treated ants exhibited 304 low aggression levels, similar to tetracycline-treated pairs. We found a significant 305 effect of the interaction faecal droplet*treatment (χ 2 =7.57, df=2, p<0.05), but not 306 diet treatment*nestmate ( χ 2 =3.36, df=2, p=0.18; Figure 2b,  and RhiAcro1 decreased in tetracycline-and moderately in sucrose-treated 325 individuals, while wSinvictaB was largely unaffected by diet (Figures 3, S5a). The NMDS ordination showed that the gut bacterial communities of tetracycline-336 treated individuals before and after faecal droplet feeding were not significantly 337 different. Contrarily, the gut bacterial communities of sugar-treated individuals 338 were closer to those of untreated workers (from original fungus gardens) after 339 faecal droplet feeding (Round 1), suggesting a shift towards the original 340 communities ( Figure S5). Interestingly, the gut bacterial samples of sugar-treated (nestmates or non-nestmates) they were fed on (PERMANOVA, F 1,43 =23.17, 343 p<0.001 and F 1,43 =12.10, p<0.001, respectively and F 1,43 =5.04, p=0.004 for their 344 interaction), whereas there was no such effect in the tetracycline-treated group 345 ( Figure S5). Tetracycline-treated ants undergoing faecal droplet feeding showed 346 an increase of all OTUs but EntAcro2 and RhiAcro1, whose levels were further 347 reduced; sucrose-treated ants showed an increase of all gut bacterial taxa 348 examined (Figure 3)

Regression of bacterial changes and aggression 353
To identify which of the OTUs best explains aggression, and since the high 354 variation both in aggression and gut microbiota composition precluded us from 355 comparing mean effects, we regressed the observed aggression between 356 nestmate test pairs on differences in abundance of their gut bacteria (Figure 4). 357 Foremost of all, this analysis confirmed that more aggression was observed 358 when nestmate pairs differed more in their gut microbial community (1500 MiSeq 359 OTUs, using Bray-Curtis distances between pairs, binomial GLMM with 360 'distance' and 'round' as fixed variables and 'colony' as random variable, z=4.91, 361 treatments (because there is a highly significant correlation between the two data 402 sets), but not to such an extent that they accurately reflect odour differences 403 between individuals that may lead to (a lack of) recognition. 404

Experimental treatment affects gut microbial communities 406
The gut microbial communities of tetracycline-treated ants underwent the most 407 substantial deviation from those of untreated nestmates (two taxa decreased 408 strongly, three slightly and one was not affected; Figure 3 After faecal droplets administration, nestmate dyadic encounters revealed 502 aggression levels only moderately (and non-significantly) higher than those of 503 relatively longer separation of the experimental ant groups (three instead than 505 two weeks in different petri dishes), the lack of significant differences between 506 rounds is consistent with only partial restoration of microbial gut communities. In 507 non-nestmate aggression assays, pairs of tetracycline-treated and tetracycline-508 vs. sucrose-treated ants again showed low aggression. In contrast, aggression 509 was high in sucrose-treated pairs when interacting individuals were fed with 510 faecal droplets from different colonies. This implies that, although bacterial 511 communities were only partially restored, colony-specific faecal droplets did 512 affect the cues determining behavioural outcomes of the social interactions. This 513 partial restoration also rules out the possibility of changes in behaviour due a 514 direct effect of tetracycline, which is known to impair mitochondrial function and 515 thus potentially could induce confounding effects (64). 516 517

518
In this study, we seek to take an integrative approach to explore the role of gut 519 microbial symbionts in the social dynamics of a eusocial organism. Remodelling 520 of the ant gut microbiota produced effects on both the chemicals ants display as 521 socially-relevant recognition signals and their resulting behaviours. Our findings 522 suggest that the observed effects mostly depend on two bacterial taxa, the 523 previously identified major gut symbionts of leafcutting ants EntAcro1 and 524 RhiAcro1. Further research will be needed to address the mechanisms 525 underlying the link between these symbionts and behavioural modifications. 526 Either the altered microbial communities may result in chemical profiles that 527 cause individuals to look more like non-nestmates, or microbiota-remodelled ants to prevent the spread of infections to optimize colony health and efficiency. 530 Regardless, our findings provide evidence that gut bacterial symbionts may be 531 involved in kin recognition by contributing to shape ant CHC signatures, with 532 implications for our understanding of social insect-symbiont evolution. 533 whereas black font shows comparisons across treatment categories (e.g. trials 557 between sucrose-treated nestmates compared to trials between tetracycline-treated nestmates). In non-nestmate encounters, sucrose-treated individuals fed 559 with faecal droplets from different colonies (≠) show the highest aggression 560 levels, whereas non-nestmates fed with faecal droplets from the same colonies 561 (=) exhibit low aggression. NM = nestmates; nNM = non-nestmates; FD= Faecal 562 droplets. See Table S2 for details on statistical comparisons among treatment 563 groups. 564