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Use of the Internal Transcribed Spacer (ITS) Regions to Examine Symbiont Divergence and as a Diagnostic Tool for Sodalis-Related Bacteria.

Snyder AK, Adkins KZ, Rio RV - Insects (2011)

Bottom Line: Additionally, we propose an ITS region as a diagnostic marker for the identification of additional Sodalis-allied symbionts in the field.These results expand our knowledge of informative genome regions to assess genetic divergence since splitting from the last common ancestor, of this versatile insect symbiont clade that have become increasingly recognized as valuable towards our understanding of the evolution of symbiosis.These facultative and recently associated symbionts may provide a novel source of traits adaptable to the dynamic ecologies encountered by diverse host backgrounds.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, West Virginia University, Morgantown, WV 26506, USA. asnyde19@mix.wvu.edu.

ABSTRACT
Bacteria excel in most ecological niches, including insect symbioses. A cluster of bacterial symbionts, established within a broad range of insects, share high 16S rRNA similarities with the secondary symbiont of the tsetse fly (Diptera: Glossinidae), Sodalis glossinidius. Although 16S rRNA has proven informative towards characterization of this clade, the gene is insufficient for examining recent divergence due to selective constraints. Here, we assess the application of the internal transcribed spacer (ITS) regions, specifically the ITS(glu) and ITS(ala,ile), used in conjunction with 16S rRNA to enhance the phylogenetic resolution of Sodalis-allied bacteria. The 16S rRNA + ITS regions of Sodalis and allied bacteria demonstrated significant divergence and were robust towards phylogenetic resolution. A monophyletic clade of Sodalis isolates from tsetse species, distinct from other Enterobacteriaceae, was consistently observed suggesting diversification due to host adaptation. In contrast, the phylogenetic distribution of symbionts isolated from hippoboscid flies and various Hemiptera and Coleoptera were intertwined suggesting either horizontal transfer or a recent establishment from an environmental source. Lineage splitting of Sodalis-allied bacteria into symbiotic and free-living sister groups was also observed. Additionally, we propose an ITS region as a diagnostic marker for the identification of additional Sodalis-allied symbionts in the field. These results expand our knowledge of informative genome regions to assess genetic divergence since splitting from the last common ancestor, of this versatile insect symbiont clade that have become increasingly recognized as valuable towards our understanding of the evolution of symbiosis. These facultative and recently associated symbionts may provide a novel source of traits adaptable to the dynamic ecologies encountered by diverse host backgrounds.

No MeSH data available.


Organization of the two Sodalis ITS1 variants. Locations of the Sodalis-allied symbiont specific PCR primers (Sg16SF and SgITSR) and ITS sequencing primers (ITSfor and ITSrev) are indicated.
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f4-insects-02-00515: Organization of the two Sodalis ITS1 variants. Locations of the Sodalis-allied symbiont specific PCR primers (Sg16SF and SgITSR) and ITS sequencing primers (ITSfor and ITSrev) are indicated.

Mentions: To amplify the ITS1 regions, primers were designed to the 3′ region of the Sodalis 16S rRNA gene (NC_007712; ITSfor: 5′-GGA GTG GGT TGC AAA AGA AG-3′) and the 5′ region of the 23S rRNA gene (ITSrev: 5′-CCA CCG TGT ACG CTT AGT CA-3′) (Figure S1) using the default Primer3 algorithm [39]. DNA samples were subjected to PCR amplification in 50 μL reactions consisting of 1.25 U GoTaq Flexi DNA Polymerase (Promega, Madison, WI, USA), 4 mM MgCl2, 1× Green GoTaq Flexi Buffer, and 0.2 mM dNTPs and primers. Amplification conditions consisted of 3 min initial denaturation at 95 °C, followed by 34 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1.5 min, with a final elongation at 72 °C for 10 min. Negative controls were included in all reactions.


Use of the Internal Transcribed Spacer (ITS) Regions to Examine Symbiont Divergence and as a Diagnostic Tool for Sodalis-Related Bacteria.

Snyder AK, Adkins KZ, Rio RV - Insects (2011)

Organization of the two Sodalis ITS1 variants. Locations of the Sodalis-allied symbiont specific PCR primers (Sg16SF and SgITSR) and ITS sequencing primers (ITSfor and ITSrev) are indicated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4553445&req=5

f4-insects-02-00515: Organization of the two Sodalis ITS1 variants. Locations of the Sodalis-allied symbiont specific PCR primers (Sg16SF and SgITSR) and ITS sequencing primers (ITSfor and ITSrev) are indicated.
Mentions: To amplify the ITS1 regions, primers were designed to the 3′ region of the Sodalis 16S rRNA gene (NC_007712; ITSfor: 5′-GGA GTG GGT TGC AAA AGA AG-3′) and the 5′ region of the 23S rRNA gene (ITSrev: 5′-CCA CCG TGT ACG CTT AGT CA-3′) (Figure S1) using the default Primer3 algorithm [39]. DNA samples were subjected to PCR amplification in 50 μL reactions consisting of 1.25 U GoTaq Flexi DNA Polymerase (Promega, Madison, WI, USA), 4 mM MgCl2, 1× Green GoTaq Flexi Buffer, and 0.2 mM dNTPs and primers. Amplification conditions consisted of 3 min initial denaturation at 95 °C, followed by 34 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 1.5 min, with a final elongation at 72 °C for 10 min. Negative controls were included in all reactions.

Bottom Line: Additionally, we propose an ITS region as a diagnostic marker for the identification of additional Sodalis-allied symbionts in the field.These results expand our knowledge of informative genome regions to assess genetic divergence since splitting from the last common ancestor, of this versatile insect symbiont clade that have become increasingly recognized as valuable towards our understanding of the evolution of symbiosis.These facultative and recently associated symbionts may provide a novel source of traits adaptable to the dynamic ecologies encountered by diverse host backgrounds.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, West Virginia University, Morgantown, WV 26506, USA. asnyde19@mix.wvu.edu.

ABSTRACT
Bacteria excel in most ecological niches, including insect symbioses. A cluster of bacterial symbionts, established within a broad range of insects, share high 16S rRNA similarities with the secondary symbiont of the tsetse fly (Diptera: Glossinidae), Sodalis glossinidius. Although 16S rRNA has proven informative towards characterization of this clade, the gene is insufficient for examining recent divergence due to selective constraints. Here, we assess the application of the internal transcribed spacer (ITS) regions, specifically the ITS(glu) and ITS(ala,ile), used in conjunction with 16S rRNA to enhance the phylogenetic resolution of Sodalis-allied bacteria. The 16S rRNA + ITS regions of Sodalis and allied bacteria demonstrated significant divergence and were robust towards phylogenetic resolution. A monophyletic clade of Sodalis isolates from tsetse species, distinct from other Enterobacteriaceae, was consistently observed suggesting diversification due to host adaptation. In contrast, the phylogenetic distribution of symbionts isolated from hippoboscid flies and various Hemiptera and Coleoptera were intertwined suggesting either horizontal transfer or a recent establishment from an environmental source. Lineage splitting of Sodalis-allied bacteria into symbiotic and free-living sister groups was also observed. Additionally, we propose an ITS region as a diagnostic marker for the identification of additional Sodalis-allied symbionts in the field. These results expand our knowledge of informative genome regions to assess genetic divergence since splitting from the last common ancestor, of this versatile insect symbiont clade that have become increasingly recognized as valuable towards our understanding of the evolution of symbiosis. These facultative and recently associated symbionts may provide a novel source of traits adaptable to the dynamic ecologies encountered by diverse host backgrounds.

No MeSH data available.