Endomicrobia in termite guts: symbionts within a symbiont (Phylogeny, cospeciation with host flagellates, and preliminary genome analysis)
Wakako Ikeda-Ohtsubo
"Endomicrobia" are intracellular symbionts of termite gut flagellates that represent a distinct lineage in the novel bacterial phylum Termite Group I (TG-1). The evolutionary history of "Endomicrobia" with respect to their symbiosis with host flagellates was investigated using phylogenetic analyses and in situ identification based on small-subunit ribosomal RNA (SSU rRNA) sequences.
By analyzing SSU rRNA sequences extracted from manually separated flagellate cells, "Endomicrobia" were shown to be widely distributed among termite gut flagellates. Symbionts originating from the same genus of flagellates invariably formed a host-specific monophyletic cluster in the phylogenetic tree. Their intracellular location in the cytoplasm was confirmed by fluorescent in situ hybridization (FISH) using oligonucleotide probes designed specifically for each symbiont and for the host. The phylogeny of "Endomicrobia" and host flagellates belonging to the parabasalid genus Trichonympha was investigated further in detail. SSU rRNA trees of the symbiont and the host exhibited overall congruence, which suggested cospeciation. Pairwise distance analysis and FISH corroborated the phylogenetic evidence, and these results allowed the construction of evolutionary scenarios for the divergence of "Endomicrobia" and their acquisition by flagellate hosts.
"Endomicrobia" share their intracellular habitat with other bacterial symbionts. Bacterial SSU rRNA sequences originating from Trichonympha flagellates of Incisitermes marginipennis and Zootermopsis nevadensis revealed the occurrence of several distinct phylogenetic groups, representing Treponema spp., "Endomicrobia", δ-Proteobacteria, Bacteroidetes, and Mycoplasmatales. The Proteobacteria symbionts were shown to densely colonize the surface and the cytoplasm of the flagellates in high abundance.
Since no pure cultures of "Endomicrobia" or their host flagellates are available, a method for the physical enrichment of "Endomicrobia" was established to gain more insights into the nature of these symbionts. "Candidatus Endomicrobium trichonymphae" (CET), the symbiont of Trichonympha flagellates, was selected as representative and enriched from gut contents of Z. nevadensis. High-molecular-weight DNA extracted from the enrichment is currently used for genome sequencing at the DOE Joint Genome Institute. A recently assembled 80-kb contig of CET revealed first insights into its metabolism, including hexuronate metabolism and the possible formation of H2.
"Endomicrobia" are also present in the gut of the wood-feeding cockroach Cryptocercus punctulatus, which is considered to share a common ancestor with termites. Analysis of SSU rRNA sequences obtained from whole-gut DNA of this cockroach revealed the phylogenetic positions of six lineages (morphotypes) of parabasalid flagellates. Sequences obtained from manually isolated flagellates, which have long been assigned to the genus Trichonympha, turned out to be a previously undescribed lineage of Parabasalia. Since this new lineage may represent one of the earliest branches of parabasalid flagellates, the recovery of “Endomicrobia" sequences also from this flagellate underscores the presence of these endosymbionts already in the flagellates of the hypothetical dictyopteran ancestor of termites and cockroaches.
The results of this study collectively document that "Endomicrobia" are prevalent and persistent endosymbionts of termite gut flagellates. This study also provides a better understanding of the phylogenetic properties of their biotic environment, i.e., the host flagellates and the cohabiting bacteria, which may help to explain the functional roles of "Endomicrobia" and their symbiotic interactions.
Philipps-Universität Marburg
Life sciences
urn:nbn:de:hebis:04-z2008-00748
opus:1905
https://doi.org/10.17192/z2008.0074
Philipps-Universität Marburg
Phylogenetische Analyse
ths
Brune Andreas (Prof. Dr.)
2008-02-20
ppn:195210441
urn:nbn:de:hebis:04-z2008-00748
Phylogenetic analysis
"Endomicrobia" are intracellular symbionts of termite gut flagellates that represent a distinct lineage in the novel bacterial phylum Termite Group I (TG-1). The evolutionary history of "Endomicrobia" with respect to their symbiosis with host flagellates was investigated using phylogenetic analyses and in situ identification based on small-subunit ribosomal RNA (SSU rRNA) sequences.
By analyzing SSU rRNA sequences extracted from manually separated flagellate cells, "Endomicrobia" were shown to be widely distributed among termite gut flagellates. Symbionts originating from the same genus of flagellates invariably formed a host-specific monophyletic cluster in the phylogenetic tree. Their intracellular location in the cytoplasm was confirmed by fluorescent in situ hybridization (FISH) using oligonucleotide probes designed specifically for each symbiont and for the host. The phylogeny of "Endomicrobia" and host flagellates belonging to the parabasalid genus Trichonympha was investigated further in detail. SSU rRNA trees of the symbiont and the host exhibited overall congruence, which suggested cospeciation. Pairwise distance analysis and FISH corroborated the phylogenetic evidence, and these results allowed the construction of evolutionary scenarios for the divergence of "Endomicrobia" and their acquisition by flagellate hosts.
"Endomicrobia" share their intracellular habitat with other bacterial symbionts. Bacterial SSU rRNA sequences originating from Trichonympha flagellates of Incisitermes marginipennis and Zootermopsis nevadensis revealed the occurrence of several distinct phylogenetic groups, representing Treponema spp., "Endomicrobia", δ-Proteobacteria, Bacteroidetes, and Mycoplasmatales. The Proteobacteria symbionts were shown to densely colonize the surface and the cytoplasm of the flagellates in high abundance.
Since no pure cultures of "Endomicrobia" or their host flagellates are available, a method for the physical enrichment of "Endomicrobia" was established to gain more insights into the nature of these symbionts. "Candidatus Endomicrobium trichonymphae" (CET), the symbiont of Trichonympha flagellates, was selected as representative and enriched from gut contents of Z. nevadensis. High-molecular-weight DNA extracted from the enrichment is currently used for genome sequencing at the DOE Joint Genome Institute. A recently assembled 80-kb contig of CET revealed first insights into its metabolism, including hexuronate metabolism and the possible formation of H2.
"Endomicrobia" are also present in the gut of the wood-feeding cockroach Cryptocercus punctulatus, which is considered to share a common ancestor with termites. Analysis of SSU rRNA sequences obtained from whole-gut DNA of this cockroach revealed the phylogenetic positions of six lineages (morphotypes) of parabasalid flagellates. Sequences obtained from manually isolated flagellates, which have long been assigned to the genus Trichonympha, turned out to be a previously undescribed lineage of Parabasalia. Since this new lineage may represent one of the earliest branches of parabasalid flagellates, the recovery of “Endomicrobia" sequences also from this flagellate underscores the presence of these endosymbionts already in the flagellates of the hypothetical dictyopteran ancestor of termites and cockroaches.
The results of this study collectively document that "Endomicrobia" are prevalent and persistent endosymbionts of termite gut flagellates. This study also provides a better understanding of the phylogenetic properties of their biotic environment, i.e., the host flagellates and the cohabiting bacteria, which may help to explain the functional roles of "Endomicrobia" and their symbiotic interactions.
Endosymbionts
Flagellates
Flagellaten
Cospeciation
117
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Endomicrobia in termite guts: symbionts within a symbiont (Phylogeny, cospeciation with host flagellates, and preliminary genome analysis)
Fachbereich Biologie
Termites
doctoralThesis
2007
2007-12-20
Endomicrobia in Termitendarm: Symbionten von Symbionten (Stammesgeschichte, Kospeziation mit den Wirtsflagellaten und vorläufige Genomanalyse)
Publikationsserver der Universitätsbibliothek Marburg
Universitätsbibliothek Marburg
"Endomicrobia" sind intrazelluläre Symbionten, die eine eigene Linie im
bakteriellen Phylum "Termite Group I" (TG-1) darstellen. Sie konnten bisher
nur in Darmflagellaten von Termiten nachgewiesen werden. In dieser Arbeit
wurde die Stammesgeschichte (Phylogenie) der "Endomicrobia" anhand von
Analysen der Sequenzvariabilität und in situ Hybridisierung der rRNA der
kleinen ribosomalen Untereinheit (SSU rRNA) untersucht.
Das für die SSU rRNA von "Endomicrobia" kodierende Gen konnte in allen
manuell sortierten Flagellatensuspensionen nachgewiesen werden. Die
phylogenetische Analyse der Gensequenzen ergab, dass aus der gleichen
Flagellatengattung stammenden Symbionten jeweils eine monophyletische Gruppe
im Stammbaum der TG-1 bildeten. Mittels "Fluoreszenz-in-situ-Hybridisierung"
(FISH) der SSU rRNA mit spezifischen Oligonukleotid-Sonden konnten die
Symbionten in den jeweiligen Wirtszellen lokalisiert werden.
Die Phylogenie der "Endomicrobia" und deren Wirtsflagellaten wurden im
weiteren bei Trichonympha-Arten (Parabasalida) im Detail untersucht. Die
phylogenetische Analyse zeigte eine klare Kongruenz der Stammbäume von Wirt
und Symbiont, was auf Kospeziation der Partner hinweist.
Paarweise-Distanz-Analyse und FISH bestätigten dieses Ergebnis und
ermöglichten die Entwicklung eines evolutionären Szenarios, welches die
Aufspaltung der "Endomicrobia" mit ihrer Aufnahme in Flagellaten erklärt.
"Endomicrobia" teilen sich das intrazelluläre Habitat mit anderen
bakteriellen Symbionten. Die Untersuchung der bakteriellen SSU-rRNA-Gene aus
den Trichonympha-Arten im Darm der Termitenarten Incisitermes marginipennis
und Zootermopsis nevadensis ergab die Anwesenheit verschiedener weiterer
Bakterien aus den Stämmen der Proteobacteria, Bacteroidetes und
Mycoplasmatales. Vertreter des Unterstammes der δ-Proteobacteria bildeten
dichte Rasen auf der Oberfläche der Flagellaten, waren aber auch in deren
Zytoplasma vertreten.
Da keine Reinkulturen von "Endomicrobia" oder derer Wirtsflagellaten
existieren, wurde für weitere Untersuchungen eine Methode zur physikalischen
Anreicherung der Symbionten erarbeitet. Als Zielorganismus wurde „Candidatus
Endomicrobium trichonymphae“ (CET), der Symbiont von Trichonympha-Spezies
gewählt und aus Z. nevadensis angereichert. Die hochmolekulare DNA aus einer
solchen Anreicherung wird momentan am JGI sequenziert, um die Genomsequenz
des Symbionten zu erhalten. Die bisher assemblierten Sequenzen (80 kb)
ergaben bereits erste Hinweise auf den Stoffwechsel von CET (z.B. den
Hexuronat-Metabolismus).
"Endomicrobia" wurden auch im Darm der holzfressenden Schabe Cryptocercus
punctulatus gefunden, die einen gemeinsamen Vorfahren mit den Termiten hat.
Die Analyse der 16S rRNA-Gensequenzen aus Darmextrakten der Schabe ergab
sechs Linien von Parabasalia Flagellaten, die bisher stammesgeschichtlich
nicht zugeordnet waren. Das Vorkommen von "Endomicrobia" in einer dieser
Linien, die einen sehr frühen Zweig der parabasaliden Flagellaten darstellt,
unterstützt zudem die Theorie, dass Termiten und Schaben einen gemeinsamen
Vorfahren haben.
Die Ergebnisse dieser Arbeit zeigen, dass "Endomicrobia" häufige und stetige
Endosymbionten von Termitendarmflagellaten sind. Sie vermitteln außerdem ein
besseres Verständnis ihrer biotischen Umgebung, und bilden damit eine
Grundlage für die weitere Erforschung der funktionellen Interaktionen von
"Endomicrobia" mit dem Wirtsflagellaten und anderen symbiotischen Bakterien.
Wakako Ikeda-Ohtsubo
opus:1905
Kospeziation
2011-08-10
monograph
Life sciences
Biowissenschaften, Biologie
English
Endosymbionten
Ecological and evolutionary drivers of microbial community structure in termite guts
https://doi.org/10.17192/z2008.0074
Termiten
Biologie
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