Diversity and function of the bacterial community associated with ants of the genus Pseudomyrmex (Lund, 1831)

Der Ameisengattung Pseudomyrmex gehören zahlreiche Baum bewohnende Arten an, die z. T. unabhängig voneinander enge Mutualismen mit verschiedenen myrmekophytischen Pflanzen eingegangen sind. Dabei verteidigen die Ameisen ihre Wirtspflanze gegen Fraßfeinde. Wie war es möglich, dass sich innerhalb dieser Gruppe so oft Generalisten mit breitem Nahrungsspektrum zu z. T. hoch spezialisierten Pflanzenameisen mit rein pflanzlicher Ernährungsweise entwickeln konnten? Oftmals liegt der Schlüssel zum Verständnis spezialisierter phytophager Insekten in ihrem Mutualismus mit Mikroorganismen, die ihnen helfen, ihre Nahrung zu verdauen und diese durch Syntheseleistungen mit essentiellen Nährstoffen wie Aminosäuren und Sekundärstoffen wie z. B. Vitaminen anzureichern. Ist es daher möglich, dass es symbiotische Bakterien sind, die es anzestral räuberischen und generalistischen Ameisen ermöglicht haben, hoch spezialisierte Vegetarier zu werden? Als konkrete Problemstellungen ergaben sich hieraus folgende Fragen: Welche bakteriellen Gemeinschaften lassen sich in Vertretern der Gattung Pseudomyrmex überhaupt finden? Unterscheidet sich die Bakterienausstattung zwischen den Mutualisten und den parasitischen Ameisen? Was ist die Funktion der Bakterien? Ist die mit den Ameisen assoziierte Bakteriengemeinschaft vielleicht sogar in der Lage, atmosphärischen Stickstoff zu fixieren? Um einen möglichst umfassenden Überblick über die mit Pseudomyrmex assoziierten Bakterien zu gewinnen, wurde eine Variante der tRFLP-Methode (Terminaler Restriktionsfragmentlängenpolymorphismus) unter Nutzung des Computerprogramms TReFID (Terminal Restriction Fragment Identifying Program) eingesetzt (Rösch und Bothe 2005, Rösch et al. 2006). Dabei werden nach einer spezifischen PCR zur Amplifikation des prokaryotischen 16S rRNA-Gens mit fluorochrommarkierten Primern die Amplifikate einem genau definierten Restriktionsverdau mit 13 verschiedenen Enzymen unterzogen. Die entstehenden terminalen markierten Fragmente werden von TReFID automatisch mit einer Prokaryoten-Datenbank abgeglichen und all diejenigen Mikroorganismen identifiziert, deren tRF-Muster (Restriktionsfragmentmuster) mit bekannten Datenbankeinträgen übereinstimmen. Diese Methode erlaubt eine qualitative Analyse der Diversität einer Bakteriengemeinschaft. Die tRFLP-Methode wurde auf drei Arten der Gattung Pseudomyrmex angewandt, die drei unterschiedliche Lebensweisen repräsentieren: mutualistische Pflanzenameise, opportunistischer Ameisenpflanzenparasit und generalistische Ameise. Ferner wurden die mutualistischen Akazien der Akazienameisen und einige myrmekophile Akazienarten untersucht und alternative mikrobiologische Verfahren (Klonierung und Kultivierung) als Vergleichsmöglichkeit zur tRFLP-Methode herangezogen. Die mit den Ameisen assoziierte bakterielle Gesellschaft konnte dabei erfolgreich charakterisiert werden. Es konnte gezeigt werden, dass die mit den Ameisen assoziierte Bakteriengemeinschaft sehr viel diverser war, als bisherige Untersuchungen an anderen verwandten Ameisengattungen dies hätte vermuten lassen. Dabei dominierten die Gruppen α-, β- und γ-Proteobacteria, Actinobacteria, Spirochaetes, Sphingobacteria, Bacilli, Clostridia, Flavobacteria und Bacteroidetes, die 90 % der Diversität der tRF-Muster darstellten. Zudem ähnelte die bakterielle Gemeinschaft sehr jener der Termiten. Da es sich bei der Biozönose des Termitendarms um eine Anpassung an defizitäre, einseitige Kost (Cellulose) handelt, konnte vermutet werden, dass eine derartige Funktion auch bei den Ameisen vorliegt. Es konnten viele Mikroorganismen nachgewiesen werden, die auf fermentative Prozesse und Stickstoff-Recycling im Darm der Ameisen schließen lassen. Die Beobachtung einer aktiven Ethin-Reduktion deutete auf aktive Nitrogenase und damit Stickstoff-Fixierung bei P. gracilis und P. salvini hin, dies konnte jedoch bislang mittels 15N2-Isotopenanalyse nicht bestätigt werden. Dafür ergaben die Daten der 15N2-Isotopenanalyse der Freilandproben von P. salvini, dass sich anders als bislang vermutet, diese Ameisenart in situ rein pflanzlich ernährt. Weitere Versuche insbesondere mit P. ferrugineus, P. gracilis aber auch weiteren arborealen und bekanntermaßen auf pflanzliche Kost, extrafloraler Nektar oder Honigtau spezialisierten Ameisenarten mit der 15N2-Methode erscheinen als erforderlich, um endgültig Stickstoff-Fixierungskapazitäten von Ameisen und deren assoziierte Mikroorganismen aufdecken oder widerlegen zu können.Ant-plant mutualisms represent a particular form of indirect defence of plants, since ants provide the defensive effect for plants against herbivorous animals. The neotropical arboreal ant genus Pseudomyrmex comprises many specialised inhabitants of myrmecophytes. Astonishingly, many of those ant species that are closely associated with plants have evolved independently from each other. How was the evolution of highly specialised vegetarian ant species from omnivorous ancestors possible? I hypothesise that microbial symbionts play a key role in the evolution of such ants, as it has already been described for insect herbivores. For example, the bacterial symbionts of Hemiptera and Isoptera synthesise amino acids, vitamins and digestive enzymes and might facilitate or impede the adaptive evolutionary diversification of the hosts. Hence, I addressed the following questions: How is the bacterial community composed that is associated to Pseudomyrmex? Are there any differences in the bacterial community between generalist, parasitic and mutualistic ants? What is the function of the bacterial community? Is the bacterial community associated with Pseudomyrmex capable of nitrogen fixation? In silico analysis of terminal restriction fragments (tRF) of fluorochrome-labelled PCR products utilising the assignment tool TReFID (Rösch und Bothe 2005, Rösch et al. 2006) was used for the gross characterisation of the bacterial community in workers and larvae of Pseudomyrmex salvini (generalist), P. gracilis (facultative parasite of the Acacia-Pseudomyrmex mutualism) and P. ferrugineus (obligate mutualist). In short, TReFID applies in silico analysis of terminal restriction fragments (tRF) obtained from digestions of fluorochrome-labelled PCR products of the 16S rRNA gene with multiple restriction enzymes. The current database consists of 22.239 entries of the NCBI database (www.ncbi.nlm.nih.gov). The advantage of this approach is the ability to characterise the bacterial diversity in a cost-effective and time-saving manner. The tRFLP approach is based on fragment length analysis and stands in contrast to clone libraries with hundreds of single sequencing steps. After obtaining an overview of the bacterial diversity of a sample with the tRFLP approach it was possible to focus on bacteria of interest. The presence of different bacterial genera was independently confirmed via PCR and cultivation approaches. The bacterial community associated with Pseudomyrmex turned out to be much more diverse than expected based on earlier studies on related ants. In all three ant species, the bacterial community was dominated by α-, β- and γ-Proteobacteria, Actinobacteria, Spirochaetes, Sphingobacteria, Bacilli, Clostridia, Flavobacteria and Bacteroidetes, which made up 90 % of tRF pattern diversity. The community structure was similar to the prokaryotic community of the hindgut of termites, another insect group that is adapted to a highly specialised diet. This similarity might indicate an adaptation of the investigated ants to a nutrient-deficient diet. Several of the bacteria identified in the present study might play a role in fermentation and N-recycling processes in the insect gut. Indeed, the observation of an active acetylene reduction indicated the presence of functioning nitrogenase in P. gracilis and P. salvini. However, consecutive stable isotope assays did not confirm an assimilation of atmospheric nitrogen by the ant organism, while data obtained on natural distributions of 15N2 indicated a strictly vegetarian diet of P. salvini under natural conditions. Further 15N2 experiments with P. ferrugineus, P. gracilis and other arboreal ants that are known to feed as direct (EFN, food bodies) or indirect (honeydew) phytophages will be required to investigate the possibility and capacity of N-fixation of ants and their associated microorganisms

Host Plant Use by Competing Acacia-Ants: Mutualists Monopolize While Parasites Share Hosts

Protective ant-plant mutualisms that are exploited by non-defending parasitic ants represent prominent model systems for ecology and evolutionary biology. The mutualist Pseudomyrmex ferrugineus is an obligate plant-ant and fully depends on acacias for nesting space and food. The parasite Pseudomyrmex gracilis facultatively nests on acacias and uses host-derived food rewards but also external food sources. Integrative analyses of genetic microsatellite data, cuticular hydrocarbons and behavioral assays showed that an individual acacia might be inhabited by the workers of several P. gracilis queens, whereas one P. ferrugineus colony monopolizes one or more host trees. Despite these differences in social organization, neither of the species exhibited aggressive behavior among conspecific workers sharing a tree regardless of their relatedness. This lack of aggression corresponds to the high similarity of cuticular hydrocarbon profiles among ants living on the same tree. Host sharing by unrelated colonies, or the presence of several queens in a single colony are discussed as strategies by which parasite colonies could achieve the observed social organization. We argue that in ecological terms, the non-aggressive behavior of non-sibling P. gracilis workers — regardless of the route to achieve this social structure — enables this species to efficiently occupy and exploit a host plant. By contrast, single large and long-lived colonies of the mutualist P. ferrugineus monopolize individual host plants and defend them aggressively against invaders from other trees. Our findings highlight the necessity for using several methods in combination to fully understand how differing life history strategies affect social organization in ants

Bacterial Associates of Arboreal Ants and Their Putative Functions in an Obligate Ant-Plant Mutualism▿ †

Bacterial communities are highly diverse and have great ecological importance. In the present study, we used an in silico analysis of terminal restriction fragments (tRF) to characterize the bacterial community of the plant ant Pseudomyrmex ferrugineus. This species is an obligate inhabitant of Acacia myrmecophytes and feeds exclusively on plant-derived food sources. Ants are the dominant insect group in tropical rain forests. Associations of ants with microbes, which contribute particularly to the ants’ nitrogen nutrition, could allow these insects to live on mostly or entirely plant-based diets and could thus contribute to the explanation of the high abundances that are reached by tropical ants. We found tRF patterns representing at least 30 prokaryotic taxa, of which the Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Planctomycetes, Proteobacteria, and Spirochaetes comprised 93%. Because most bacterial taxa were found in all ant-derived samples studied and because the bacteria detected on the ants’ host plant revealed little overlap with this community, we regard our results as reliably representing the bacterial community that is associated with P. ferrugineus. Genera with a likely function as ant symbionts were Burkholderia, Pantoea, Weissella, and several members of the Enterobacteriaceae. The presence of these and various other groups was confirmed via independent PCR and cultivation approaches. Many of the bacteria that we detected belong to purportedly N-fixing taxa. Bacteria may represent important further partners in ant-plant mutualisms, and their influences on ant nutrition can contribute to the extraordinary abundance and evolutionary success of tropical arboreal ants

Representative cuticular hydrocarbon profile of the mutualist <i>Pseudomyrmex ferrugineus</i> (A) and the parasite <i>Pseudomyrmex gracilis</i> (B).

<p>The profile of <i>P. gracilis</i> consisted of 26 hydrocarbons and the profile of <i>P. ferrugineus</i> of 18 hydrocarbons. Peak numbers correspond to the compounds as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037691#pone.0037691.s003" target="_blank">Table S2</a>, ‘imp.’ denotes impurity, ‘ster.’ denotes steroid. Pooled extracts from 10 ant individuals.</p

Aggressiveness of ants after replacement within plots.

<p>Each plot consisted of eight trees. Five ant individuals from one tree (rows) were individually placed on another tree (columns) and the encounter with an ant individual from the tree it was placed on was observed. Behavior was either classified as aggressive (black background), neutral (white background) or ambiguous (grey shades with darker shades indicating a higher proportion of aggressive encounters). Numbers of ants that reacted aggressive or neutral are indicated in the boxes (aggressive/neutral).</p

Genetic diversity measures within each study plot of the parasite <i>Pseudomyrmex gracilis</i> in South Mexico as obtained from female genotypes.

<p>n denotes the total number of female individuals for each plot; <i>N</i>_A denotes observed number of alleles found at each locus from each plot; <i>H</i>_E = expected heterozygosity; <i>H</i>_O = observed heterozygosity;</p>*<p>significant deviation according to HW-Probability test (<i>P</i><0.05).</p

Relatedness (mean ± SD; R-value) among the workers sampled from each acacia.

<p>Mutualist refers to <i>Pseudomyrmex ferrugineus</i>, Parasite to <i>P. gracilis</i>.</p>‘*’<p>indicates significant deviation from 0.75 (as among full sisters in monogynous colonies) according to T-test. n denotes number of individuals included from each acacia.</p

Spatial distribution of the acacia plants from which ants were sampled in each plot.

<p>Figures are based on GPS data.</p

Discriminant analyses of ant hydrocarbon profiles.

<p>Hydrocarbon profiles of all individuals that were collected from the eight acacias from each plot are plotted according to their distribution along with the first and second root extracted. Percentages on axes indicate the variance explained by the respective root.</p