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.

NJ-Phylogram, gaps included

Uncorrected p-distances, bootstrap values: NJ / parsimony

based on 395 nt of bacterial 16S rDNA

Nostoc commune AB113665Neisseia cinerea AY831725

Bordetella holmesii AJ239044Burkholderia cepacia AB211225

Symbiont of T. polita RT13-2Bacillus cibi AY550276

Flavobacterium mizutaii AJ438175Flavobacterium frigidarium AF162266Flexibacter sancti AB078068

Symbiont of T. allaborans RT17-3Leptospira parva AY293856Symbiont of T. allaborans RT17-6Brevibacterium mcbrellneri X93594

Symbiont of T. allaborans RT17-9Wolbachia pipientis AJ628417

Symbiont of T. allaborans RT17-2Wolbachia sp. WCR AY007551

Buchnera aphidicola AY620431Symbiont of Echinopla pallipes RE1

Blochmannia floridanus NC_005061 Symbiont of T. extenuata RT4-1

Symbiont of T. extenuata RT4-10Symbiont of T. extenuata RT4-3

Symbiont of T. allaborans RT17-1Symbiont of T. cf. allaborans RT15Yersinia pestis AF366383

Serratia liquefaciens AY253924Sodalis glossinidius AY861704

Symbiont of T. pilosa RT10-1Pantoea agglomerans AJ583011Symbiont of T. attenuata RT1-6

Camponotus floridanus midgut isolateEscherichia coli NC_000913 Klebsiella variicola AJ783916

Caulobacter sp. AJ227766Rickettsia prowazekii M21789

Rhodospirillum rubrum D30778Azorhizobium caulinodans X67221

Methylobacterium aminovorans AJ851086Afipia birgiae AF288304

Bradyrhizobium betae AY372184Rhizobium lusitanum AY738130

Rhizobium etli AY904730Rhizobium leguminosarum AY946012

Symbiont of T. attenuata RT1-5Agrobacterium tumefaciens D14500

Symbiont cf. Rhizobium of T. binghami AF459798 Bartonella chomelii AY254309

Bartonella henselae AJ223780Bartonella elizabethae L01260Bartonella sp. from Apis mellifera AY370185

Symbiont of Dolichoderus coniger RE2Symbiont of T. binghami HF3-2

Symbiont of T. attenuata RT1-7Symbiont of T. polita RT13-1Symbiont of T. attenuata RT6-3

Ochrobactrum anthropi AY917134Brucella melitensis AY513568

Ochrobactrum sp. AF028733Rhodobium orientis D30792

Mesorhizobium sp. AY332116Phyllobacterium catacumbae AY636000

Aminobacter aganoensis AJ011760Mesorhizobium loti D12791Sinorhizobium meliloti AY904728

Pseudaminobacter salicylatoxidans AJ294416Uncultured α-proteobacterium from Collembola AJ604541

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T. nigra-group

T. allaborans-group

T. pilosa

Pro

teob

acteria

unique sequences detected by cloning

Dolichoderus

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.

nigra-groupnigra-group

allaborans-groupallaborans-group

pilosa-grouppilosa-group

Eubacterial 16S-PCREubacterial 16S-PCRFig. 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.

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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. 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. 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).

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.

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).

Fig. 6 (below): Phylogeny of amplified nifH genes.

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“)

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b

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.