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 cultureindependent 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 ␥-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 ␣-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.

Something from (almost) nothing: the impact of multiple displacement amplification on microbial ecology

“…The extraction of community nucleic acids is a typical initial step, and although cell biomass is often sufficiently high to enable analysis by PCR, gene hybridization or metagenomics, this is not always the case. Because microbial ecologists study challenging environments, such as insect guts (Broderick et al, 2004), ice cores (Christner et al, 2001), permafrost (Steven et al, 2006), deep subsurface sediments (Teske, 2005) and air (Brodie et al, 2007), high-sensitivity PCR protocols have been required for the analysis of single genes. A whole genome amplification (WGA) step provides access to community DNA from these low-biomass environments.…”

The Effect of Plant Quality on Survival of Lymantria Dispar (Lepidoptera: Lymantriidae) Larvae Infected by Bacillus Thuringiensis Berliner Subsp. Kurstaki

“…This insect feeds on a wide range of plants which contain allelochemicals, such as phenolics, tannins, and terpenoids and a great number of these allelochemicals are toxic to bacteria (Broderick et al, 2004;Dillon and Dillon, 2004). In a host, selection pressure on the microbial community may be caused by toxic compounds.…”

A Pharm-Ecological Perspective of Terrestrial and Aquatic Plant-Herbivore Interactions

“…There is increasing evidence that most mammalian herbivores, and many invertebrate herbivores harbor large populations of microbes (bacteria, protozoa and fungi) in their gut that can play a variety of roles including making nutrients more available to the host animal (Broderick et al 2004;Chandler et al, 2008;Clark et al 2010;Clements et al, 2009;Janson et al, 2008;Mountfort et al, 2002;Stevens and Hume, 1998).…”

“…For example, gut microbes might provide enzymes such as cellulases, to efficiently digest plant material ). Aquatic and terrestrial herbivorous Lepidoptera larvae harbor distinct communities of bacteria in the gut despite their small body size and the rapid passage of food through gut, and these microbial communities differ depending on the plants eaten (Broderick et al, 2004;Walenciak et al, 2002). Whether these bacteria are involved in the detoxification of PSM remains to be seen.…”