Census of the bacterial community of the gypsy moth larval midgut by using culturing and culture-independent methods

Appl Environ Microbiol. 2004 Jan;70(1):293-300. doi: 10.1128/aem.70.1.293-300.2004.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Bacteria / classification
  • Bacteria / genetics
  • Bacteria / growth & development*
  • Bacteria / isolation & purification*
  • Culture Media
  • DNA, Ribosomal / analysis
  • Diet
  • Ecosystem*
  • Genes, rRNA
  • Intestines / microbiology*
  • Larva / microbiology
  • Larva / physiology
  • Lepidoptera / microbiology*
  • Lepidoptera / physiology
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Polymorphism, Restriction Fragment Length
  • RNA, Ribosomal, 16S / genetics*
  • Sequence Analysis, DNA

Substances

  • Culture Media
  • DNA, Ribosomal
  • RNA, Ribosomal, 16S