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Applied and Environmental Microbiology, December 2008, p. 7189-7196, Vol. 74, No. 23
0099-2240/08/$08.00+0     doi:10.1128/AEM.01464-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Origin and Diversity of Metabolically Active Gut Bacteria from Laboratory-Bred Larvae of Manduca sexta (Sphingidae, Lepidoptera, Insecta){triangledown}

Nicole Brinkmann, Rainer Martens, and Christoph C. Tebbe*

Institute of Biodiversity, Johann Heinrich von Thünen-Institut (vTI), Braunschweig, Germany

Received 30 June 2008/ Accepted 2 October 2008

Cultivation-independent analyses based on genetic profiling of partial bacterial 16S rRNA genes by PCR-single-strand conformation polymorphism (PCR-SSCP), reverse transcriptase (RT)-PCR-SSCP of the 16S rRNA itself, and stable isotope probing (SIP), followed by RT-PCR-SSCP, were applied to characterize the diversity of metabolically active bacteria in the larval gut of Manduca sexta bred on tobacco leaves under greenhouse conditions. For SIP, hatching larvae were fed with leaves from tobacco plants grown in a 13CO2-enriched atmosphere. Dominant SSCP bands were sequenced and phylogenetically analyzed. Only one major gut colonizer, an Enterococcus relative, was detected; it occurred in the heavy RNA fraction, demonstrating its metabolic activity, and it originated from eggs, where its metabolic activity was also indicated by rRNA-based SSCP profiles. In contrast, a Citrobacter sedlakii relative was detected on eggs by DNA-SSCP, but rRNA-SSCP and SIP-rRNA-SSCP were negative, suggesting that these bacterial cells were inactive. A Burkholderia relative was dominant and metabolically active on the tobacco leaves but inactive inside the gut, where it was also quantitatively reduced, as suggested by lower band intensities in the DNA-based SSCP profiles. SIP-RNA-SSCP detected another metabolically active gut bacterium (Enterobacter sp.) and more bacteria in the light RNA fraction, indicating low or no metabolic activity of the latter inside the gut. We conclude that the larval gut supported only a low diversity of metabolically active bacteria.

* Corresponding author. Mailing address: Institut für Biodiversität, Johann Heinrich von Thünen-Institut (vTI), Bundesallee 50, 38116 Braunschweig, Germany. Phone: 49 531 596 2553. Fax: 49 531 596 2599. E-mail: christoph.tebbe{at}vti.bund.de

{triangledown} Published ahead of print on 10 October 2008.

Applied and Environmental Microbiology, December 2008, p. 7189-7196, Vol. 74, No. 23
0099-2240/08/$08.00+0     doi:10.1128/AEM.01464-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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