Can the presence of bacterial endosymbionts explain the abundance of arboreal ants?

1. Eubacterial 16S-PCR allaborans-group nigra-group pilosa-group Can the presence of bacterial endosymbionts explain the abundance of arboreal ants? Sascha Stoll1, Roy Gross1, Heike Feldhaar2 University of Würzburg, Germany 1Department of Microbiology, 2Department of Behavioral Physiology and Sociobiology Introduction. Ants (Hymenoptera: Formicidae) are hyperdominant in tree crowns of tropical rain forests. Recent studies using stable isotopes (Blüthgen et al. 2003) have shown that some arboreal ant species can achieve their abundance by feeding principally as herbivores i.e. foraging on homopteran exudates and nectar. Since these food resources are scarce in nitrogen it has been speculated that symbiotic microorganisms of homopterans or ants themselves play a key role in nutritional upgrading by recycling waste nitrogen or fixing atmospheric dinitrogen. Screening for symbiont diversity. We conducted a comparative study of the bacterial gut microflora of several species from three different clades of the arboreal ant genus Tetraponera (Stoll et al. 2006). For one of these clades (T. nigra group) Billen and Buschinger (2000) have described a gut pouch with a sophisticated ultrastructure that is densely filled with yet unidentified bacteria (Fig. 1). Species of other clades of the genus lacked such a pouch. In our study symbiont diversity was surveyed by 16S rDNA - TGGE (Fig. 2), cloning and phylogenetic analyses. Fig. 1: worker of Tetraponera attenuata, a species belonging to the nigra group. The small picture shows a gut preparation of this species. The red arrow marks the bacteria-filled gut pouch between the midgut and hindgut. Fluorescent in situ hybridization (FISH). The location of the Bartonella-like symbiont of T. attenuata in the gut pouch was confirmed by FISH with eubacterial and Rhizobiales-specific Cy3-labled oligonucleotide probes (Fig. 4). Fig. 2: Temperature Gradient Gel Electrophoresis (TGGE) of universally primed bacterial 16S rDNA PCR fragments. In addition to electrophoresis DNA fragments of the same size are separated according to their sequence-specific melting behavior in an underlying temperature gradient, yielding a bacterial community fingerprint of the examined ant guts. Tetraponera species from the same phylogenetic clade reveal a similar symbiont pattern while clearly differing from the other clades’ bacterial communities. Samples of interest are subsequently cloned, sequenced and analyzed with phylogenetic models. - b a + Fig. 4: FISH of T. attenuata gut pouches Dissected pouches were squashed on microscope slides and analyzed by FISH. The pouches harbor masses of branched, Y-shaped bacteria (see Fig. 4b for detailed view). Host group specific symbiont pattern in Tetraponera ants. For all 12 colonies examined belonging to four different species of the nigra-group we detected a bacterial symbiont forming a monophyletic cluster within the order Rhizobiales (α-Proteobacteria) with similarities to Bartonella and Mesorhizobium and evidence for coevolution with their hosts. In all species examined lacking the pouch we found prokaryotes that are closely related to endosymbionts of other arthropods, e.g. Sodalis (S-endosymbiont of tsetse flies) or Enterobacter agglomerans, a bacterium that is known to fix nitrogen in termites (Fig. 3). Fig 5 (left): FISH of a Dolichoderus sp hindgut In spite of close phylogenetic relations to the pouch-bacteria of Tetraponera, the main symbionts of Dolichoderus ants show a different morphology and are located in the ants’ hindgut. Fig. 3: 16S rDNA phylogram of bacterial symbionts found in this survey. The sequences indicate close coevolution with the ant hosts of the different clades. Intriguingly, a close relative of the Bartonella-like symbiont of all Tetraponera species from the nigra-group was also found in several species of the genus Dolichoderus which belong to a different subfamily, but suffer from similar nutritional restraints. Nostoc commune AB113665 96/95 Neisseia cinerea AY831725 100/100 Bordetella holmesii AJ239044 Burkholderia cepacia AB211225 100/100 Symbiont of T. polita RT13-2 Bacillus cibi AY550276 100/100 Flavobacterium mizutaii AJ438175 Fig. 6 (below): Phylogeny of amplified nifH genes. Flavobacterium frigidarium AF162266 -/63 51/- Flexibacter sancti AB078068 100/100 Symbiont of T. allaborans RT17-3 88/- Leptospira parva AY293856 100/100 Symbiont of T. allaborans RT17-6 Brevibacterium mcbrellneri X93594 unique sequences detected by cloning 61/- Symbiont of T. allaborans RT17-9 100/100 Wolbachia pipientis AJ628417 95/69 Symbiont of T. allaborans RT17-2 Wolbachia sp. WCR AY007551 Buchnera aphidicola AY620431 -/72 98/66 Symbiont of Echinopla pallipes RE1 Blochmannia floridanus NC_005061 87/90 98/100 Symbiont of T. extenuata RT4-1 100/62 Symbiont of T. extenuata RT4-10 T. allaborans-group Evidence for dinitrogen fixation. In several colonies from all Tetraponera-clades we have found preliminary evidence for a nitrogen-fixing microbiota by successful amplification of nifH (dinitrogenase subunit) suggesting an important and general role of these gut microorganisms in nutritional upgrading in these arboreal ants (Fig. 6). Symbiont of T. extenuata RT4-3 54/- Symbiont of T. allaborans RT17-1 86/65 g Symbiont of T. cf. allaborans RT15 75/88 Yersinia pestis AF366383 Serratia liquefaciens AY253924 57/- Sodalis glossinidiusAY861704 -/59 76/69 100/99 Symbiont of T. pilosa RT10-1 T. pilosa Pantoea agglomeransAJ583011 55/- 88/- Symbiont of T. attenuata RT1-6 Camponotus floridanus midgut isolate 63/- Escherichia coli NC_000913 85/88 Klebsiella variicola AJ783916 Caulobacter sp. AJ227766 Rickettsia prowazekii M21789 Rhodospirillum rubrum D30778 55/- 54/61 Azorhizobium caulinodans X67221 Methylobacterium aminovorans AJ851086 100/100 60/- Afipia birgiae AF288304 Bradyrhizobium betae AY372184 90/81 Rhizobium lusitanum AY738130 100/93 Rhizobium etli AY904730 Rhizobium leguminosarum AY946012 64/70 Symbiont of T. attenuata RT1-5 a Proteobacteria 98/57 Agrobacterium tumefaciens D14500 Symbiont cf. Rhizobium of T. binghami AF459798 70/- 57/85 Bartonella chomelii AY254309 Dolichoderus Bartonella henselae AJ223780 62/- Bartonella elizabethae L01260 58/- Bartonella sp. from Apis melliferaAY370185 Symbiont of Dolichoderus coniger RE2 Symbiont of T. binghami HF3-2 -/70 100/100 T. nigra-group Symbiont of T. attenuata RT1-7 Symbiont of T. polita RT13-1 68/- Symbiont of T. attenuata RT6-3 54/- Ochrobactrum anthropi AY917134 98/58 Brucella melitensis AY513568 Ochrobactrum sp. AF028733 Rhodobium orientis D30792 Mesorhizobium sp. AY332116 Phyllobacterium catacumbae AY636000 64/- Aminobacter aganoensis AJ011760 Mesorhizobium loti D12791 Sinorhizobium meliloti AY904728 56/- Pseudaminobacter salicylatoxidans AJ294416 75/- Uncultured α-proteobacterium from Collembola AJ604541 0.01 substitutions/site NJ-Phylogram, gaps included Uncorrected p-distances, bootstrap values: NJ / parsimony based on 395 nt of bacterial 16S rDNA Conclusion. The presence of a host group specific, possibly nitrogen-fixing gut microbiota in ant genera from nutrient-poor habitats could help to explain the superabundance of these animals and their evolutionary success. Functional studies will help us to understand the mechanisms of these symbioses. References: Billen, J. and A. Buschinger. 2000. Morphology and ultrastructure of a specialized bacterial pouch in the digestive tract of Tetraponera ants (Formicidae, Pseudomyrmecinae). Arthropod. Struct.Dev. 29:259-266.Blüthgen, N., G. Gebauer and K. Fiedler. 2003. Disentangling a rainforest food web using stable isotopes: dietary diversity in a species-rich ant community. Oecologia 137:426-435.Stoll S., J. Gadau, R. Gross, H. Feldhaar. 2006. Bacterial microbiota associated with ants of the genus Tetraponera. Biol. J. Linn. Soc. (accepted) This work is funded by the Elitenetzwerk Bayern (BayEFG) and the DFG (SFB 567 “Mechanisms of interspecific interactions“)