Folie 1

1
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 (a-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
Neisseia cinerea AY831725
96/95
100/100
Bordetella holmesii AJ239044
Burkholderia cepacia AB211225
100/100
Symbiont of T. polita RT13-2
Bacillus cibi AY550276
Flavobacterium mizutaii AJ438175
100/100
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
Symbiont of T. allaborans RT17-9
61/-
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
Sodalis glossinidius AY861704
57/-
-/59
76/69
100/99
Symbiont of T. pilosa RT10-1
T. pilosa
Pantoea agglomerans AJ583011
88/-
55/-
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/-
Azorhizobium caulinodans X67221
54/61
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
Symbiont of T. attenuata RT1-5
64/70
a
Proteobacteria
98/57
Agrobacterium tumefaciens D14500
Symbiont cf. Rhizobium of T. binghami AF459798
70/-
57/85
Bartonella chomelii AY254309
Bartonella henselae AJ223780
Dolichoderus
62/-
Bartonella elizabethae L01260
58/-
Bartonella sp. from Apis mellifera AY370185
Symbiont of Dolichoderus coniger RE2
Symbiont of T. binghami HF3-2
100/100
-/70
T. nigra-group
Symbiont of T. attenuata RT1-7
Symbiont of T. polita RT13-1
68/-
Symbiont of T. attenuata RT6-3
Ochrobactrum anthropi AY917134
54/-
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 a-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. 29259-266.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 137426-435.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)