Mechanismen, die Pflanzen-Ameisen Interaktionen, ermöglichen: Acacia-Pseudomyrmex als Modellsystem

Mutualisms are interactions among different species that lead to net fitness benefits for all partners involved. In plant-ant mutualisms, plants provide to ants an array of rewards, such as extrafloral nectar (EFN), food bodies, or nesting space. Ants are attracted, or completely nourished, by plant-derived food rewards and serve as a means of indirect defence of plants against herbivores. Although these mutualisms can become very specific, the rewards traded among mutualist partners may also be attractive for non-mutualist organisms, i.e., exploiters that make use of the host-derived rewards without reciprocating. Thus, the goal of this study was to investigate mechanisms that drive the specificity of plant-ant interactions, and that stabilize it from exploitation. The mutualism of Acacia plants with Pseudomyrmex ants was used as a model system, in which we can find different kinds of plant-ant interactions that vary in their specificity: facultative and obligate. Whereas Acacia obligate plants (myrmecophytes) secrete EFN at high quantities and constituvely, to house and nourish symbiotic ants of P. ferrugineus, facultative ones (non-myrmecophytes) secrete it only in response to damage, attracting generalist ants. These differences in plant-ant interactions make this genus Acacia highly suitable to study mechanisms that may determine species-specific interaction. Specifically, I focused my study on the chemistry of EFN (amino acids and proteins) and on the ant behaviour in terms of defence against nectar robbers, herbivores and leaf pathogens. Amino acid composition of obligate Acacia was highly specialized and adapted to the preferences and nutritive requirements of the specialised mutualist ant P. ferrugineus. Mutualist ants preferred EFN solutions that contained exactly those amino acids that were quantitatively dominating in myrmecophyte EFN. By contrast, generalist ants preferred sugar solutions with amino acids over mere sugar solutions but were not able to discriminate among different numbers or concentrations of specific amino acids, suggesting, thus, that amino acids of non-myrmecophyte EFN play an important role in the attraction but less so in the nutrition of ants. On the other hand, EFN of obligate Acacia species appeared (bio)chemically protected from microbe infestation. Bioassays demonstrated that fungal growth was inhibited in EFN of myrmecophytes. The identification of proteins in myrmecophyte nectar revealed an abundant presence of PR-proteins, such as glucanases, chitinases and thaumatin- and osmotin like proteins, of which activities were also detected in EFNs. Furthermore, the total amount of proteins was significantly higher in myrmecophyte EFN than in the EFN of non-myrmecophytes. These data, together with the observations that the protein-fraction of myrmecophyte EFN significantly inhibited the growth of various fungi, suggests that nectar proteins are associated with the protection of EFN from microbes. In parallel to these chemical adaptations on the side of the plant, symbiotic ants of P. ferrugineus, unlike the parasite P. gracilis, exhibited relevant ecological and chemical adaptations, which contribute to the specificity of the mutualism. P. ferrugineus effectively defended their host plants against herbivores and leaf bacteria and protected the EFN from nectar robbers. Nevertheless, the defensive efficiency provided by P. ferrugineus was associated with the amounts of rewards provided by the host plant: the host species that invest less in ant rewards received less defence by the symbiotic ant. Thus, P. ferrugineus tended to diminish its defensive service when it did not receive the respective pay-off from the host. On the other hand, P. ferrugineus had the capacity to induce EFN secretion by myrmecophytes, demonstrating that the host plant also can cease reward production when it does not receive the expected biotic defence. The results of the present study illustrate different chemical and ecological mechanisms that drive the specificity of the Acacia-Pseudomyrmex mutualism, thus, helping 1) to prevent the mutualism from exploitation and, 2) to stabilize the mutualist interaction.Mutualismen sind Interaktionen verschiedener Arten, bei denen ein Partner einen „Service“ erbringt, welcher von einem anderen Partner „belohnt“ wird. In Pflanzen-Ameisen Mutualismen bieten Pflanzen Ameisen Nahrung in Form von extrafloralem Nektar (EFN) sowie Futterkörperchen und in einigen Fällen auch Nistraum. Im Gegenzug verteidigen Ameisen ihre Pflanze gegen Fraßfeinde, Herbivore und Pathogene. Oft ist es für die Pflanze ein Problem, Nektarkonsumenten fernzuhalten, die keine Gegenleistung erbringen und nur ökologische Kosten verursachen, so genannte „Exploiter“. Ziel meiner Arbeit war die Untersuchung von Mechanismen, die zur Stabilität von Pflanzen-Ameisen Interaktionen führen und vor der Ausnutzung durch solche „Exploiter“ schützen. Der Mutualismus zwischen Acacia und Pseudomyrmex wurde als Modell verwendet, da verschiedene Spezifitätsgrade innerhalb des Systems auftreten. Während obligate Ameisenpflanzen, so genannte Myrmekophyten, EFN ständig in hohen Raten produzieren, um symbiotische Ameisenkolonien dauerhaft zu ernähren, produzieren die so genannten myrmekophilen Akazienarten EFN erst als eine Antwort auf Herbivorie um Ameisen aus der Umgebung anzulocken. Diese unterschiedlichen Spezifitätsgrade von Pflanzen-Ameisen Interaktionen innerhalb der Gattung Acacia erlauben es, artspezifische Interaktionen zu untersuchen. Im Focus meiner Arbeit standen die Untersuchung der chemischen Komponenten des EFNs (Aminosäuren und Proteine) sowie die Untersuchung des Verhaltens von Ameisen im Hinblick auf Verteidigung gegenüber Nektarräubern, Herbivoren und Pathogenen. Die Aminosäure-Zusammensetzung der myrmekophytischen Akazien war höchst speziell und angepasst an die Präferenzen und Nährstoffbedürfnisse der mutualistischen Ameisen P. ferrugineus. Mutualistische Ameisen bevorzugten genau solche EFN-Lösungen, welche die vier quantitativ dominierenden Aminosäuren im Myrmecophyten-EFN enthielten. Im Gegensatz dazu bevorzugten generalistische Ameisen Zuckerlösungen mit Aminosäuren vor reine Zuckerlösungen. Die Generalisten unterschieden jedoch nicht zwischen Anzahl oder Konzentration spezifischer Aminosäuren. Diese Ergebnisse deuten darauf hin, dass der EFN der myrmekophilen Akazien eine wichtige Rolle für das Anlocken von Ameisen und weniger für deren Ernährung spielt. Weiterhin scheint der EFN der obligaten Akazien (bio)chemisch geschützt vor der Besiedlung durch Mikroben zu sein. In Bioassys war Pilzwachstum durch Myrmekophyten-EFN gehemmt. PR-Proteine (pathogenesis related) wie Glucanasen, Chitinasen, Thaumatin- und Osmotin-Proteine wurden im Myrmekophyten-EFN identifiziert und die entsprechenden Enzymaktivitäten konnten nachgewiesen werden. Zudem war die absolute Menge von Proteinen signifikant höher im EFN der Myrmekophyten als im EFN der myrmekophilen Arten. Diese Ergebnisse deuten zusammen mit der Beobachtung, dass der Proteinanteil des Myrmekophyten-EFNs das Wachstum von verschiedenen Pilzen inhibierte, auf eine Rolle der EFN-Proteine im Schutz vor Mikroben hin. Parallel zu diesen Anpassungen der Myrmekophyten auf biochemischer Ebene zeigten mutualistischen Ameisen der Art P. ferrugineus — im Gegensatz zu der parasitischen Art P. gracilis — wichtige ökologische and chemische Anpassunge, welche eine hohe Spezifität des Mutualismus bewirken. Pseudomyrmex ferrugineus Ameisen verteidigten die Wirtspflanzen effektiv gegen Herbivores und Pathogene und schützten den EFN vor Nektarräubern. Dennoch war die Effizienz der Verteidigung durch P. ferrugineus mit der Menge an Belohnung, welche durch die Pflanze bereit gestellt wurde, verbunden: Wirtspflanzen, die wenig in Belohnungen für die Ameisen investierten, wurden auch weniger effizient durch Ameisen verteidigt. Pseudomyrmex ferrugineus verminderte also die Verteidigung, wenn die Ameisen nicht entsprechende Belohnungen von der Pflanze erhielten. Andererseits war die mutualistische Ameisenart in der Lage die EFN-Sekretion durch Myrmeckophyten zu induzieren. Das zeigt, dass die Wirtspflanzen die Nektarproduktion verringern, wenn sie nicht die erwartene Verteidigung der Ameisen erhalten. Insgesamt konnte ich in meiner Arbeit verschiedene chemische und ökologische Mechanismen identifizieren, die die Spezifizität des Acacia-Pseudomyrmex Mutualismus aufrechterhalten, die den Mutualismus vor Ausbeutung schützen und die das mutualistische System stabilisieren

Increased host investment in extrafloral nectar (EFN) improves the efficiency of a mutualistic defensive service

Extrafloral nectar (EFN) plays an important role as plant indirect defence through the attraction of defending ants. Like all rewards produced in the context of a mutualism, however, EFN is in danger of being exploited by non-ant consumers that do not defend the plant against herbivores. Here we asked whether plants, by investing more in EFN, can improve their indirect defence, or rather increase the risk of losing this investment to EFN thieves. We used the obligate plant-ant Acacia-Pseudomyrmex system and examined experimentally in the field during the dry and the rainy seasons how variations in EFN secretion are related to (i) ant activity, to (ii) the ant-mediated defence against herbivores and (iii) the exploitation of EFN by non-ant consumers. Extrafloral investment enhanced ant recruitment and was positively related to the ant mediated defence against herbivores. The ant-mediated protection from exploiters also increased in proportion to the nectar sugar concentration. Although the daily peak of EFN production coincided with the highest activity of EFN thieves, Pseudomyrmex ferrugineus ants protected this resource effectively from exploiters. Nevertheless, the defensive effects by ants differed among seasons. During the dry season, plants grew slower and secreted more EFN than in the rainy season, and thus, experienced a higher level of ant-mediated indirect defence. Our results show that an increased plant investment in an indirect defence trait can improve the resulting defensive service against both herbivores and exploiters. EFN secretion by obligate ant-plants represents a defensive trait for which the level of investment correlates positively with the beneficial effects obtained

Genome-scale analyses of health-promoting bacteria: probiogenomics

The human body is colonized by an enormous population of bacteria (microbiota) that provides the host with coding capacity and metabolic activities. Among the human gut microbiota are health-promoting indigenous species (probiotic bacteria) that are commonly consumed as live dietary supplements. Recent genomics-based studies (probiogenomics) are starting to provide insights into how probiotic bacteria sense and adapt to the gastrointestinal tract environment. In this Review, we discuss the application of probiogenomics in the elucidation of the molecular basis of probiosis using the well-recognized model probiotic bacteria genera Bifidobacterium and Lactobacillus as examples

Conservation of thermospermine synthase activity in vascular and non-vascular plants

[EN] In plants, the only confirmed function for thermospermine is regulating xylem cells maturation. However, genes putatively encoding thermospermine synthases have been identified in the genomes of both vascular and non-vascular plants. Here, we verify the activity of the thermospermine synthase genes and the presence of thermospermine in vascular and non-vascular land plants as well as in the aquatic plant Chlamydomonas reinhardtii. In addition, we provide information about differential content of thermospermine in diverse organs at different developmental stages in some vascular species that suggest that, although the major role of thermospermine in vascular plants is likely to be xylem development, other potential roles in development and/or responses to stress conditions could be associated to such polyamine. In summary, our results in vascular and non-vascular species indicate that the capacity to synthesize thermospermine is conserved throughout the entire plant kingdom.This work in the laboratories was funded by grants BFU2016-80621-P and BIO2016-79147-R of the Spanish Ministry of Economy, Industry and Competitiveness. AS-G and JH-G are recipients of Fellowships of the Spanish Ministry of Science, Innovation and Universities BES-2017-080387 and of the Spanish Ministry of Education, Culture and Sport FPU15/01756, respectively. JA holds a Ramón y Cajal Contract RYC-2014-15752.Solé-Gil, A.; Hernández-García, J.; López-Gresa, MP.; Blazquez Rodriguez, MA.; Agusti Feliu, J. (2019). Conservation of thermospermine synthase activity in vascular and non-vascular plants. Frontiers in Plant Science. 10:1-10. https://doi.org/10.3389/fpls.2019.00663S11010Ashton, N. W., & Cove, D. J. (1977). 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Neotropical ant-plant Triplaris americana attracts Pseudomyrmex mordax ant queens during seedling stages

The association between the myrmecophyte Triplaris and ants of the genus Pseudomyrmex is an often-reported example of mutualism in the Neotropics. The ants colonize the hollow stems of their hosts, and in exchange, the plants benefit from a reduced degree of herbivory. The previous studies have shown that workers can discriminate their host from other plants, including a closely related species. Little is known about how queens locate their host during the colonization process, but it has been suggested that host recognition is mediated by volatiles. Since queens of Pseudomyrmex mordax colonize their hosts during the seedling stage, we hypothesized that queens would discriminate leaves of seedlings from adult plants. To evaluate our hypothesis, we used a two-sided olfactometer, to test the preference of queens towards different leaf and plant ages of Triplaris americana. Virgin queens of Pseudomyrmex mordax preferred seedlings over adult plants, as well as plant leaves over empty controls, showing no discrimination for leaf age. Our results suggest that the volatiles virgin queens recognize are either produced or are more abundant at the early growing stage of the host when colonization is crucial for the host's survival. © 2017, The Author(s)

Mode of action, chemistry and defensive efficacy of the osmeterium in the caterpillar <i>Battus polydamas archidamas</i>

Chemical secretions are one of the main defensive mechanisms in insects. The osmeterium is a unique organ in larvae of Papilionidae (Lepidoptera), which is everted upon disturbance, secreting odoriferous volatiles. Here, using larvae of the specialized butterfly Battus polydamas archidamas (Papilionidae: Troidini), we aimed to understand the mode of action of the osmeterium, the chemical composition and origin of the secretion, as well as its defensive efficiency against a natural predator. We described osmeterium's morphology, ultramorphology, structure, ultrastructure, and chemistry. Additionally, behavioral assays of the osmeterial secretion against a predator were developed. We showed that the osmeterium is composed of tubular arms (made up by epidermal cells) and of two ellipsoid glands, which possess a secretory function. The eversion and retraction of the osmeterium are dependent on the internal pressure generated by the hemolymph, and by longitudinal muscles that connect the abdomen with the apex of the osmeterium. Germacrene A was the main compound present in the secretion. Minor monoterpenes (sabinene and ss-pinene) and sesquiterpenes ((E)-beta-caryophyllene, selina-3,7(11)-diene, and other some unidentified compounds) were also detected. Only sesquiterpenes (with the exception of (E)-beta-caryophyllene) are likely to be synthesized in the osmeterium-associated glands. Furthermore, the osmeterial secretion proved to deter predatory ants. Our results suggest that the osmeterium, besides serving as an aposematic warning for enemies, is an efficient chemical defense, with its own synthesis of irritant volatiles