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Applied and Environmental Microbiology, January 2004, p. 293-300, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.293-300.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Census of the Bacterial Community of the Gypsy Moth Larval Midgut by Using Culturing and Culture-Independent Methods

Nichole A. Broderick,1,2,3* Kenneth F. Raffa,1 Robert M. Goodman,2,3,4 and Jo Handelsman2,3

Departments of Entomology,1 Plant Pathology,2 the Microbiology Doctoral Training Program,3 the Gaylord Nelson Institute for Environmental Studies, University of Wisconsin, Madison, Wisconsin 537064

Received 18 March 2003/ Accepted 22 September 2003

Little is known about bacteria associated with Lepidoptera, the large group of mostly phytophagous insects comprising the moths and butterflies. We inventoried the larval midgut bacteria of a polyphagous foliivore, the gypsy moth (Lymantria dispar L.), whose gut is highly alkaline, by using traditional culturing and culture-independent methods. We also examined the effects of diet on microbial composition. Analysis of individual third-instar larvae revealed a high degree of similarity of microbial composition among insects fed on the same diet. DNA sequence analysis indicated that most of the PCR-amplified 16S rRNA genes belong to the {gamma}-Proteobacteria and low G+C gram-positive divisions and that the cultured members represented more than half of the phylotypes identified. Less frequently detected taxa included members of the {alpha}-Proteobacterium, Actinobacterium, and Cytophaga/Flexibacter/Bacteroides divisions. The 16S rRNA gene sequences from 7 of the 15 cultured organisms and 8 of the 9 sequences identified by PCR amplification diverged from previously reported bacterial sequences. The microbial composition of midguts differed substantially among larvae feeding on a sterilized artificial diet, aspen, larch, white oak, or willow. 16S rRNA analysis of cultured isolates indicated that an Enterococcus species and culture-independent analysis indicated that an Entbacter sp. were both present in all larvae, regardless of the feeding substrate; the sequences of these two phylotypes varied less than 1% among individual insects. These results provide the first comprehensive description of the microbial diversity of a lepidopteran midgut and demonstrate that the plant species in the diet influences the composition of the gut bacterial community.

* Corresponding author. Mailing address: Department of Entomology, University of Wisconsin—Madison, 1630 Linden Dr., 345 Russell Laboratories, Madison, WI 53705. Phone: (608) 262-8735. Fax: (608) 262-5289. E-mail: nab{at}entomology.wisc.edu.

Applied and Environmental Microbiology, January 2004, p. 293-300, Vol. 70, No. 1
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.1.293-300.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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