Reflex bleeding is an effective defensive mechanism against predators. When attacked, some insects emit hemolymph, which coagulates, quickly entangling their aggressor. Bleeding occurs at weak intersegmental membranes or through dedicated organs, which can be associated or not with glandular cells.
Here, we describe the behavior and morphological structures involved in reflex bleeding in the larvae of the ladybird, Diomus thoracicus, which are intranidal parasites of the ant Wasmannia auropunctata. The larvae are tolerated by the ants thanks to odor mimicry, but some rare aggressive ant behaviors were observed that trigger reflex bleeding both at a pair of thoracic tubercles and a pair of posterodorsal abdominal humps. No glandular structure was found in association with these emission points, which suggests that the material emitted was hemolymph only. A 3D reconstruction suggested that reflex bleeding seems to be controlled by muscles whose contraction increases the internal hydrostatic pressure and pushes the hemolymph into a funnel-like structure with an opening to the outside. In D. thoracicus, the morphological structures involved in reflex bleeding are among the most complex and prominent described to date.

1. Land-use changes frequently lead to major changes in the composition and diversity of organisms. A reduction in the range of strategies enabling organisms to survive in a given environment and changes in the average trait values of species may potentially be associated with variations in species’ number and identity.
2. We investigated the variation in ant taxonomic composition and morphological trait diversity along a land-use gradient in French Guiana. We measured 13 core ant morphological traits on all species sampled. We then selected the set of five traits that best captured changes along the land-use gradient. Potential effects of the variation in morphological trait diversity and average values were evaluated by examining morphological traits individually as well as in combination.
3. We found that variation in taxonomic diversity was unrelated to the plot-level morphospace. Conversely, a significant shift in taxonomic composition was accompanied by changes in the average values of community traits along the studied gradient, examined both individually and in combination.
4. We argue that morphological trait values may be related to the success of different species in surviving in a given environment and, therefore, are indicative of the taxonomic turnover in ants along the land-use gradient. Nevertheless, in contradiction with theoretical expectations, the morphospace is only slightly affected by habitat filtering and loosely impacted by taxonomic changes. Examining the sensitivity of the morphospace to abiotic and biotic factors and how it reflects varying ecological pressures for species is thus of the utmost importance.

Background and Aims
The plant Hirtella physophora, the ant Allomerus decemarticulatus and a fungus, Trimmatostroma sp., form a tripartite association. The ants manipulate both the plant trichomes and the fungus to build galleries under the stems of their host plant used to capture prey. In addition to its structural role, the fungus also improves nutrient uptake by the host plant. But it still remains unclear whether the fungus plays an indirect or a direct role in transferring nutrients to the plant. This study aimed to trace the transfer of N from the fungus to the plant’s stem tissue.

Methods
Optical microscopy and transmission electron microscopy (TEM) were used to investigate the presence of fungal hyphae in the stem tissues. Then, a ¹⁵N-labelling experiment was combined with a nanoscale secondary-ion mass spectrometry (NanoSIMS 50) isotopic imaging approach to trace the movement of added ¹⁵N from the fungus to plant tissues.

Key Results
The TEM images clearly showed hyphae inside the stem tissue in the cellular compartment. Also, fungal hyphae were seen perforating the wall of the parenchyma cell. The ¹⁵N provisioning of the fungus in the galleries resulted in significant enrichment of the ¹⁵N signature of the plant’s leaves 1 d after the ¹⁵N-labelling solution was deposited on the fungus-bearing trap. Finally, NanoSIMS imaging proved that nitrogen was transferred biotrophically from the fungus to the stem tissue.

Conclusions
This study provides evidence that the fungi are connected endophytically to an ant–plant system and actively transfer nitrogen from ¹⁵N-labelling solution to the plant’s stem tissues. Overall, this study underlines how complex the trophic structure of ant–plant interactions is due to the presence of the fungus and provides insight into the possibly important nutritional aspects and tradeoffs involved in myrmecophyte–ant mutualisms.

The construction process and use of galleries by Azteca brevis (Myrmicinae: Dolichoderinae) inhabiting Tetrathylacium macrophyllum (Salicaceae) were compared with Allomerus decemarticulatus (Myrmicinae: Solenopsidini) galleries on Hirtella physophora (Chrysobalanaceae). Though the two ant species are phylogenetically distant, the gallery structure seems to be surprisingly similar and structurally convergent: both are pierced with numerous holes and both ant species use Chaetothyrialean fungi to strengthen the gallery walls. Al. decemarticulatus is known to use the galleries for prey capture and whether this is also the case for Az. brevis was tested in field experiments. We placed Atta workers as potential prey/threat on the galleries and recorded the behaviour of both ant species. We found considerable behavioural differences between them: Al. decemarticulatus was quicker and more efficient at capture than was Az. brevis. While most Atta workers were captured after the first 5 min by Al. decemarticulatus, significantly fewer were captured by Az. brevis even after 20 min. Moreover, the captured Atta were sometimes simply discarded and not taken to the nest by Az. brevis. As a consequence, the major function of the galleries built by Az. brevis may, therefore, be defense against intruders in contrast to Al. decemarticulatus which uses them mainly for prey capture. This may be due to a higher need for protein in Al. decemarticulatus compared to coccid-raising Az. brevis.

Ants have evolved venoms rich in peptides and proteins used for predation, defense, and communication. However, they remain extremely understudied due to the minimal amount of venom secreted by each ant. The present study investigated the differences in the proteome and peptidome of the venom from the bullet ant, Paraponera clavata. Venom samples were collected from a single colony either by manual venom gland dissection or by electrical stimulation and were compared using proteomic methods. Venom proteins were separated by 2D-PAGE and identified by nanoLC-ESI-QTOF MS/ MS. Venom peptides were initially separated using C18 reversed-phase high-performance liquid chromatography, then analyzed by MALDI-TOF MS. The proteomic analysis revealed numerous proteins that could be assigned a biological function (total 94), mainly as toxins, or roles in cell regulation and transport. This investigation found that ca. 73% of the proteins were common to venoms collected by the two methods. The peptidomic analysis revealed a large number of peptides (total 309) but with <20% shared by the two collection methods. There was also a marked difference between venoms obtained by venom gland dissection from different ant colonies. These findings demonstrate the rich composition and variability of P. clavata venom.

Species engaged in multiple, simultaneous mutualisms are subject to trade- offs in their mutualistic investment if the traits involved in each interaction are overlapping, which can lead to conflicts and affect the longevity of these associations. We investigate this issue via a tripartite mutualism involving an ant plant, two competing ant species and a fungus the ants cultivate to build galleries under the stems of their host plant to capture insect prey. The use of the galleries represents an innovative prey capture strategy compared with the more typical strategy of foraging on leaves. However, because of a limited worker force in their colonies, the prey capture behaviour of the ants results in a trade-off between plant protection (i.e. the ants patrol the foliage and attack intruders including herbivores) and ambushing prey in the galleries, which has a cascading effect on the fitness of all of the partners. The quantification of partners’ traits and effects showed that the two ant species differed in their mutualistic investment. Less investment in the galleries (i.e. in fungal cultivation) translated into more benefits for the plant in terms of less herbivory and higher growth rates and vice versa. However, the greater vegetative growth of the plants did not produce a positive fitness effect for the better mutualistic ant species in terms of colony size and production of sexuals nor was the mutualist compensated by the wider dispersal of its queens. As a consequence, although the better ant mutualist is the one that provides more benefits to its host plant, its lower host–plant exploitation does not give this ant species a competitive advantage. The local coexistence of the ant species is thus fleeting and should eventually lead to the exclusion of the less competitive species.