Isolation of Polymer-Degrading Bacteria and Characterization of the Hindgut Bacterial Community from the Detritus-Feeding Larvae of Tipula abdominalis (Diptera: Tipulidae)

Abstract

The Tipula abdominalis larval hindgut microbial community presumably facilitates digestion of the lignocellulosic diet. The microbial community was investigated through characterization of bacterial isolates and analysis of 16S rRNA gene clone libraries. This initial study revealed novel bacteria and provides a framework for future studies of this symbiosis.Insects are the largest taxonomic group of animals on earth. Although a few thorough studies have shown that insects host an environment with high microbial diversity (3-5, 9, 11, 17, 24), less than 1% of described insect species have been examined for microorganisms (10). Tipula abdominalis is an aquatic crane fly ubiquitous in aquatic riparian environments. T. abdominalis larvae are shredders, a functional feeding group of insects that consume coarse particulate organic matter, primarily leaf litter. In small riparian stream ecosystems, leaf litter comprises the majority of carbon and energy inputs (26); however, many organisms are unable to degrade this lignocellulosic material, which is difficult to digest due to highly structured plant polysaccharide polymers (cellulose, hemicellulose, and lignin) and which has low nutritional value due to a high C/N ratio (15). By converting lignocellulose into a form that other organisms can use, T. abdominalis larvae influence the bioavailability of carbon and energy within the ecosystem.The larva itself is not capable of tissue-level synthesis of cellulolytic enzymes (22), and it was proposed that the larvae benefit nutritionally from microbially mediated digestion of leaf lignocellulose, providing simple fermentation products which can be used by the larvae (13). Scanning electron microscopy studies demonstrated a dense and morphologically diverse microbial community in the hindgut of T. abdominalis larvae (12). This microbial community was investigated for phylogenetic diversity and enzymatic activity towards model plant polymer substrates.Larva collection and dissection. Larvae were collected from 2nd-order streams (26) in Michigan. Hindguts were extracted and transferred to a reduced buffered salt solution (BSS) (14).

Lignocellulolytic enzymes and bacteria associated with the digestive tracts of Stenochironomus (Diptera: Chironomidae) larvae

“…For example, a larger proportion of cultivable isolates of γ-proteobacteria have also been reported in different species of beetles (Park et al, 2007). At the genus level, Bacillus and Pseudomonas are recognized for their ability to degrade biopolymers (Gilbert and Hazlewood, 1993) and are common in the digestive tracts of saproxylic insects, such as tipulids, termites, and cerambycid beetles (Moore, 1972;Wenzel et al, 2002;Cook et al, 2007;Park et al, 2007). In addition to these bacteria, Sphingobium yanoikuyae was demonstrated in the present study to be present in both types of larvae analyzed.…”

“…Species of associated cellulolytic bacteria are well established in terrestrial insects (e.g., Wenzel et al, 2002;Delalibera Jr. et al, 2005). However, the use of molecular methods has only recently identified bacteria related to aquatic insects, particularly in the larvae of the genus Tipula (Diptera: Tipulidae) (Cook et al, 2007).…”

Mining diversity of the natural biorefinery housed within Tipula abdominalis larvae for use in an industrial biorefinery for production of lignocellulosic ethanol

“…Analysis of T. abdominalis hindgut bacteria using 16S rRNA gene libraries revealed a phylogenically diverse community (Cook et al , 2007). From a total of 322 clones, 163 phylotypes (operational taxonomic units sharing ≥ 99% sequence similarity), were identified and evaluated for similarity to other ribosomal RNA (rRNA) genes from clones and isolated bacteria using database comparisons.…”

“…Clostridia and Bacteroidetes clones were the only classes found in all four libraries. Using methods similar to those described (Cook et al , 2007) Clostridia and Bacteroidetes clones were compared to one another, as well as previously described uncultured and cultured bacteria, at varying percent sequence similarity. Clones were more similar to one another than to previously described sequences, and more similar to uncultured than cultured bacteria (Fig.…”

Klugiella xanthotipulae gen. nov., sp. nov., a novel member of the family Microbacteriaceae

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Strain 44C3T was isolated from the hindgut of Tipula abdominalis larvae as described by Cook et al (2007). T. abdominalis is an aquatic crane fly, larvae of which are primary shredders of leaf litter in small, riparian streams.

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