title: Census of the bacterial community of the gypsy moth larval midgut by using culturing and culture-independent methods.
abstract: 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.
authors.affiliation: Department of Entomology, University of Wisconsin, Madison, Wisconsin 53706, USA. nab@entomology.wisc.edu
authors.forename: Nichole A
authors.initials: NA
authors.lastname: Broderick
authors.forename: Kenneth F
authors.initials: KF
authors.lastname: Raffa
authors.forename: Robert M
authors.initials: RM
authors.lastname: Goodman
authors.forename: Jo
authors.initials: J
authors.lastname: Handelsman
mesh_terms: D000818:Animals; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000254:growth & development* / Q000302:isolation & purification*; D003470:Culture Media; D004275:DNA, Ribosomal / Q000032:analysis; D004032:Diet; D017753:Ecosystem; D020459:Genes, rRNA; D007422:Intestines / Q000382:microbiology*; D007814:Larva / Q000382:microbiology / Q000502:physiology; D007915:Lepidoptera / Q000382:microbiology* / Q000502:physiology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: D003470:Culture Media; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S