Microbial community structure in midgut and hindgut of the humus-feeding larva of Pachnoda ephippiata (Coleoptera: Scarabaeidae)

Abstract

The guts of soil-feeding macroinvertebrates contain a complex microbial community that is involved in the transformation of ingested soil organic matter. In a companion paper (T. Lemke, U. Stingl, M. Egert, M. W. Friedrich, and A. Brune, Appl. Environ. Microbiol. 69:6650-6658, 2003), we show that the gut of our model organism, the humivorous larva of the cetoniid beetle Pachnoda ephippiata, is characterized by strong midgut alkalinity, high concentrations of microbial fermentation products, and the presence of a diverse, yet unstudied microbial community. Here, we report on the community structure of bacteria and archaea in the midgut, hindgut, and food soil of P. ephippiata larvae, determined with cultivation-independent techniques. Clone libraries and terminal restriction fragment length polymorphism analysis of 16S rRNA genes revealed that the intestines of P. ephippiata larvae contain a complex gut microbiota that differs markedly between midgut and hindgut and that is clearly distinct from the microbiota in the food soil. The bacterial community is dominated by phylogenetic groups with a fermentative metabolism (Lactobacillales, Clostridiales, Bacillales, and Cytophaga-Flavobacterium-Bacteroides [CFB] phylum), which is corroborated by high lactate and acetate concentrations in the midgut and hindgut and by the large numbers of lactogenic and acetogenic bacteria in both gut compartments reported in the companion paper. Based on 16S rRNA gene frequencies, Actinobacteria dominate the alkaline midgut, while the hindgut is dominated by members of the CFB phylum. The archaeal community, however, is less diverse. 16S rRNA genes affiliated with mesophilic Crenarchaeota, probably stemming from the ingested soil, were most frequent in the midgut, whereas Methanobacteriaceae-related 16S rRNA genes were most frequent in the hindgut. These findings agree with the reported restriction of methanogenesis to the hindgut of Pachnoda larvae.

FIG. 1.

Phylogenetic tree showing the positions of bacterial 16S rRNA gene sequences affiliated with gram-positive bacteria, recovered from the midgut (⋄) and hindgut (•) of P. ephippiata larvae. Scale bar represents 10% sequence difference. Accession numbers of reference sequences are indicated. Species used as the outgroup were Thermus thermophilus (M26923), Fervidobacterium gondwanense (Z49117), and Thermotoga maritima (M21774). Roman numerals indicate clostridial subgroups sensu (14). Lengths of T-RFs result from in vitro digestion of clonal 16S rRNA gene amplicons with MspI; pseudo-T-RFs are marked with asterisks.

FIG. 2.

Phylogenetic tree showing the positions of bacterial 16S rRNA gene sequences affiliated with the CFB phylum, the Proteobacteria (PB), and the Planctomycetales, recovered from the midgut (⋄) and hindgut (•) of P. ephippiata larvae. Scale bar represents 10% sequence difference. Accession numbers of reference sequences are indicated. Species used as the outgroup were Thermus thermophilus (M26923), Fervidobacterium gondwanense (Z49117), and Thermotoga maritima (M21774). Lengths of T-RFs result from in vitro digestion of clonal 16S rRNA gene amplicons with MspI; pseudo-T-RFs are marked with asterisks.

FIG. 3.

Phylogenetic tree showing the positions of archaeal 16S rRNA gene sequences affiliated with Euryarchaeota and Crenarchaeota, recovered from the midgut (⋄) and hindgut (•) of P. ephippiata larvae and from the soil fed to the larvae (▵). Scale bar represents 10% sequence difference. Accession numbers of reference sequences are indicated. Species used as outgroup were Nitrospira marina (X82559), Chloroflexus aurantiacus (M34116), Holophaga foetida (X77215), Rhodothermus marinus (X77140), Streptomyces coelicolor (X60514), and Acidobacterium capsulatum (D26171). Lengths of T-RFs result from in vitro digestion of clonal 16S rRNA gene amplicons with AluI or TaqI; pseudo-T-RFs are marked with asterisks.

FIG. 4.

T-RFLP profiles of bacterial 16S rRNA genes amplified from DNA extracts of the midgut and hindgut of P. ephippiata larvae and food soil. MspI was used for restriction digestion. All major T-RFs (≥2% of total peak height; triangles), assignable minor T-RFs (dots), and peaks probably influenced by pseudo-T-RF formation (asterisks) are marked. Assignable phylogenetic groups are given in parentheses with the following abbreviations: AB, Actinobacteria; B, Bacillales; CFB, CFB phylum; C, Clostridiales, LB, Lactobacillales; P, Planctomycetales; βPB, β-Proteobacteria; n.a., not assignable. Boldface T-RFs in the soil profiles indicate major T-RFs without a corresponding major T-RF in the gut profiles.

FIG. 5.

Relative frequencies of distinct archaeal groups in the midgut, hindgut, and food soil of P. ephippiata larvae, based on composition of 16S rRNA gene clone libraries and T-RFLP analysis, with TaqI and AluI. T-RF frequencies were calculated by comparing the individual heights of assignable T-RFs to the sum of all peak heights in the electropherograms. T-RF lengths in base pairs are given in parentheses; T-RFs affected by formation of pseudo-T-RFs are marked with an asterisk (see text). C, Crenarchaeota; MB, Methanobacteriaceae; MM, Methanomicrobiales; MS, Methanosarcinales; RCII, rice cluster II; TP, Thermoplasmales, n.a., not assignable.