Axel Touchard

Venom systems are key evolutionary innovations that have contributed to the ecological success of numerous animal lineages. In ants (Formicidae), venom exhibits an exceptional diversity in both chemical composition and delivery mechanisms. Here, we combine a synthesis of current knowledge on the evolution of ant venoms with new phylogenetic comparative analyses, integrating chemical, morphological, and phylogenetic data at the genus level. Comparative analyses across Formicidae support the hypothesis that peptide-rich venoms represent the ancestral condition in ants, as they are widely distributed across basal and derived lineages. Despite this ancestral state, we identify seven independent evolutionary transitions toward non-peptidic venom, occurring in major clades including Dolichoderinae, Formicinae, Stenammini, Solenopsidini, Crematogaster , Pheidole , and fungus-growing ants. Ancestral state reconstruction and tests of correlated evolution across 114 genera show that these seven shifts away from peptide-based venom were almost irreversible and tightly coupled with the loss of stinging capacity and a dietary shift away from predation. These transitions are consistently associated with profound morphological modifications of the venom apparatus, such as reduction or complete loss of the sting, or its transformation into non-injecting structures. Chemically, these systems are dominated by alkaloids, iridoids, formic acid, esters, and terpenoids. These repeated transitions reflect major ecological and evolutionary shifts. Lineages that rely less on individual predation and more on collective foraging or plant-based diets tend to exhibit reduced reliance on injectable peptide toxins. Instead, they favor metabolite-based venoms that can be co-opted from existing biochemical pathways and deployed in diverse ecological contexts. Conversely, peptide-rich venoms are retained in lineages subjected to strong selective pressures for rapid prey immobilization or defense against vertebrate predators. Overall, ants represent a unique model for understanding venom evolution, illustrating how shifts in chemistry, morphology, and ecological strategy can drive repeated and convergent innovations. Future integrative studies combining genomics, metabolomics, and functional assays will be essential to unravel the mechanisms underlying these transitions and their role in the extraordinary diversification of ants.

Les venins sont des armes sophistiquées, utilisées par les organismes venimeux pour se défendre des prédateurs, ainsi que pour paralyser et tuer leurs proies. Mais dans la nature, le bien n’est jamais très loin du mal, les toxines venimeuses pouvant se révéler être des agents thérapeutiques efficaces. Les peptides de venins de fourmis ont donc été étudiés dans cette thèse afin de déterminer le potentiel de ces toxines pour la découverte de molécules thérapeutiques innovantes. A l’instar des autres venins d’insectes, les venins de fourmis restent peu étudiés, principalement en raison de la petite taille de ces insectes et des quantités limitées de venins disponibles. Cependant, les fourmis offrent l’avantage d’être des insectes sociaux très abondants dans tous les milieux terrestres. En collectant les venins de plusieurs individus, il est donc possible d’obtenir des quantités suffisantes de venin pour les analyses biochimiques et pharmacologiques.Afin d’assurer la reproductibilité des analyses, une identification taxonomique correcte est nécessaire. Dans cette optique, un outil de chimiotaxonomie a été développé durant cette thèse (permettant ainsi de regrouper les venins provenant de plusieurs colonies afin de compenser les faibles quantités de matériel biologique par individu ou par colonie).Ensuite, nous nous sommes intéressés aux facteurs écologiques impliqués dans la diversification des venins de fourmis. Pour cela, la toxicité et la composition des venins de fourmis ont été analysés en relation avec le polyéthisme, la spécialisation alimentaire et la spécialisation défensive.La diversité écologique des fourmis a amplement contribuée à la diversification des venins. En étudiant les venins de 82 espèces de fourmis, nous avons révélé la grande diversité structurale des toxines. Bien que la majorité des peptidomes sont composés par de petits peptides linéaires, des peptides structurés par des ponts disulfure ont été révélés dans de nombreux venins et constituent de nouvelles familles structurales de toxines.La purification de certains de ces peptides à ponts disulfure a permis leur caractérisation biochimique et l’évaluation de leur rôle biologique. Ainsi nous avons décrit un groupe de peptides neurotoxiques, baptisés les formicitoxines qui sont capables de bloquer les canaux calcium humains de type L. La commutatoxine est, quant à elle, un peptide avec un pont disulfure qui semble activer les récepteurs humains TRPV1 et TRPV3 et laisse supposer une implication dans l’induction de la douleur chez les mammifères.La grande diversité des peptides mise en évidence dans les venins, associée à la grande diversité écologique et taxonomique des fourmis, suggère que les venins de fourmis constituent un nouveau champ d’exploration prometteur pour la recherche de molécules thérapeutiques et insecticides. Les venins de fourmis s’ajoutent à la chimiothèque conséquente déjà représentée par les venins des autres animaux venimeux.

In ants, division of labour is often associated with caste‐specific adaptations. In this study, we examined the morphology of the venom apparatus (venom reservoir volume and sting length) and the composition and activity of the venom across castes (soldier, submajor and media/minor) of the army ant Eciton hamatum to investigate how caste differentiation influences venom traits. Morphometric analyses revealed that soldiers have larger venom reservoirs and longer stings, consistent with their specialised role in defending the colony against vertebrates. Integrative venom profiling using transcriptomics and mass spectrometry identified 11 venom peptides, including a glycopeptide bearing a complex N ‐Glycan. Notably, soldiers had a streamlined venom peptide composition compared with other castes. Despite this reduced complexity, soldier venom was more potent in functional assays, showing enhanced paralytic effects on insect prey and stronger pain‐inducing activity on vertebrate neurons. Chymotrypsin enzymes, more abundant in the venoms of soldier castes, may also serve a pre‐digestive role. This pattern suggests an evolutionary trade‐off favouring a limited set of highly effective components tailored to the dual function of defence and predation in soldiers.

Natural selection can drive the evolution of similar traits through convergent evolution. Short peptides identical to the vertebrate hormone bradykinin (BK) have been reported from the venoms and skin secretions of certain species of wasps (order Hymenoptera) and frogs (order Anura), respectively. In this study, we demonstrate that the genes encoding the BK-like peptides of hymenopteran venoms and anuran skin secretions do not share common ancestry with that of the vertebrate hormone but instead independently evolved multiple times from peptide toxin genes. These peptides serve a defensive function against vertebrate predators and their resemblance to BK was driven by selection for efficacy at the predators’ receptors. Our findings highlight how natural selection can drive repeated convergent evolution of similar molecules across distantly related lineages.

This study focuses on species occupying the three strata of an Amazonian rainforest: the ground and leaf litter, the understorey and the canopy. We employed only two sampling techniques: Winkler extraction for ground‐dwelling ants and direct observations for understorey and canopy species on large branches cut off by a climber. We identified 494 ant species from 10 subfamilies and 77 genera over ≈3.0 ha (Chao1 = 607 species; 95% CI: 566–670 species). Although we found fewer arboreal ants compared to approaches using insecticide fogging, this study confirms similarities between the ant diversity in Amazonian and Mesoamerican rainforests. The functional traits of these ants (i.e., diet, nest‐site preference, population size of the colony) allowed us to identify seven clusters. Cluster 1 is a “hodgepodge” grouping arboreal or ground‐dwelling species with different‐sized colonies (76 species). Cluster 2 primarily includes small colonies of ground‐nesting generalist feeders (142 species). Cluster 3 comprises all arboreal species from the understorey inhabiting myrmecophyte domatia or palm trees plus arboreal species with medium‐sized colonies (37 species). Cluster 4 includes all territorially dominant arboreal ants plus one ground‐dwelling species (21 species). All fungus‐growing species belong to Cluster 5, which also contains ground‐nesting generalist feeders and generalist predators (148 species). All doryline army ants are grouped in Cluster 6 along with one ponerine known for its nomadic behavior (15 species). Almost all specialized predators belong to Cluster 7 (55 species); however, Cluster 5 includes two ponerine species that prey exclusively on termites. Based on a nonmetric multidimensional scaling (NMDS), we confirmed that the position of these clusters corresponded fairly well to the three forest strata. Thus, analyzing functional traits enables the trophic position of most ants and their place in the vertical strata of Neotropical rainforests to be determined.

Although tropical rainforest ants are abundant and have high species richness, with their community structure well known at the canopy and ground levels, the understory is frequently neglected. To fill this gap, we looked for ants nesting in this stratum in an Amazonian rainforest and noted the occurrence of colonies in the debris accumulated between the fronds of the palm tree Astrocaryum sciophilum or associated with eight myrmecophytes (i.e., plants that shelter ant colonies in hollow structures called domatia). We analysed these plant–ant relationships by conducting a network approach looking for the degree of specialisation between partners (specialisation index), the differences in intensity between links (weighted nestedness) and the proportion of realised interactions relative to the number of potential interactions (connectance), including ground‐ and canopy‐nesting ants. This results in a highly modular pattern of interactions with one module per plant species based on the 98 ant species recorded. We showed the importance of ground‐nesting species for Astrocaryum, although 40.5% of them sheltered a colony of the arboreal Odontomachus hastatus. Among myrmecophytes, a strong specificity was noted for Hirtella physophora (Chrysobalanaceae) and Maieta guianensis (Melastomataceae) that sheltered at more than 90% Allomerus decemarticulatus and Pheidole minutula, respectively. Cordia nodosa (Boraginaceae) has an intermediary value (45.3% associated with Allomerus octoarticulatus), whereas Cecropia obtusa (Urticaceae), Duroia longiflora (Rubiaceae), Tachigali paniculata (Fabaceae) and Tococa guianensis (Melastomataceae) can shelter several ant species belonging mostly to a single genus. Tachia guianensis, considered a ‘non‐specialised’ myrmecophyte, shelters species from different genera.

Stinging ants have diversified into various ecological niches, and selective pressures may have contributed to shape the composition of their venom. To explore the drivers underlying venom variation in ants, we sampled 15 South American rainforest species and recorded a range of traits, including ecology, morphology and venom bioactivities. Principal component analysis of both morphological and venom bioactivity traits reveals that stinging ants display two functional strategies where species have evolved towards either an exclusively offensive venom or a multi-functional venom. Additionally, phylogenetic comparative analysis indicates that venom function (predatory, defensive or both) and mandible morphology correlate with venom bioactivity and volume. Further analysis of the venom biochemistry of the 15 species revealed switches between cytotoxic and neurotoxic venom compositions among species. Our study supports an evolutionary trade-off between the ability of venom to deter vertebrate predators and to paralyse insect prey which are correlated with different venom compositions and life-history strategies among Formicidae.

Stinging ants have diversified into various ecological niches, and several evolutionary drivers may have contributed to shape the composition of their venom. To comprehend the drivers underlying venom variation in ants, we selected 15 Neotropical species and recorded a range of traits, including ecology, morphology, and venom bioactivity. Principal component analysis of both morphological and venom bioactivity traits revealed that stinging ants display two functional strategies. Additionally, phylogenetic comparative analysis indicated that venom function (predatory, defensive, or both) and mandible morphology significantly correlate with venom bioactivity and amount, while pain-inducing activity trades off with insect paralysis. Further analysis of the venom biochemistry of the 15 species revealed switches between cytotoxic and neurotoxic venom compositions in some species. This study highlights the fact that ant venoms are not homogenous, and for some species, there are major shifts in venom composition associated with the diversification of venom ecological functions. Significance Venoms are under severe evolutionary pressures, exerted either on the innovation of toxins or the reduction of the metabolic cost of production (1). To reduce the metabolic costs associated with venom secretion, some venomous animals can regulate venom expenditure by metering the amount of venom injected and by switching between offensive and defensive compositions (2–2). Many ants use venom for subduing a wide range of arthropod prey, as well as for defensive purposes against invertebrates and vertebrates, but are unable to adapt venom composition to stimuli (5, 6). Consequently, the expression of venom genes directly affects the ability of ants to interact with the biotic environment, and the venom composition may be fine-tuned to the ecology of each species. A previous study showed that defensive traits in ants exhibit an evolutionary trade-off in which the presence of a sting is negatively correlated with several other defensive traits, further supporting that trade-offs in defensive traits significantly constrain trait evolution and influence species diversification in ants (7). However, the sting is not used for the same purpose depending on the ant species. Our study supports an evolutionary trade-off between the ability of venom to deter vertebrates and to paralyze insects which are correlated with different life history strategies among Formicidae.