In situ analysis of the bacterial community in the gut of the earthworm<i>Lumbricus terrestris</i>L. by whole-cell hybridization

215

153

The microbial populations in no-till agricultural soil and casts of the earthworm Lumbricus rubellus were examined by culturing and molecular methods. Clone libraries of the 16S rRNA genes were prepared from DNA isolated directly from the soil and earthworm casts. Although no single phylum dominated the soil library of 95 clones, the largest numbers of clones were from Acidobacteria (14%), Cytophagales (13%), Chloroflexi (8%), and ␥-Proteobacteria (8%). While the cast clone library of 102 clones was similar to the soil library, the abundances of several taxa were different. Representatives of the Pseudomonas genus as well as the Actinobacteria and Firmicutes increased in number, and one group of unclassified organisms found in the soil library was absent in the cast library. Likewise, soil and cast archaeal 16S rRNA gene libraries were similar, although the abundances of some groups were different. Two hundred and thirty aerobic bacteria were also isolated on general heterotrophic media from casts, burrows, and soil. The cast isolates were both phenotypically and genotypically different from the soil isolates. The cast isolates were more likely to reduce nitrate, grow on acetate and Casamino Acids, and utilize fewer sugars than the soil isolates. On the basis of their ribotypes, the cast isolates were dominated by Aeromonas spp. (28%), which were not found in the soil isolates, and other ␥-Proteobacteria (49%). In contrast, the soil isolates were mostly Actinobacteria (53%), Firmicutes (16%), and ␥-Proteobacteria (19%). Isolates obtained from the sides of earthworm burrows were not different from the soil isolates. Diversity indices for the collections of isolates as well as rRNA gene libraries indicated that the species richness and evenness were decreased in the casts from their levels in the soil. These results were consistent with a model where a large portion of the microbial population in soil passes through the gastrointestinal tract of the earthworm unchanged while representatives of some phyla increase in abundance.

Section: Discussionsupporting

confidence: 84%

Molecular and Culture-Based Analyses of Prokaryotic Communities from an Agricultural Soil and the Burrows and Casts of the EarthwormLumbricus rubellus

Furlong

Singleton²,

Coleman

et al. 2002

215

153

“…While similar numbers have been reported for soils (2), even higher percentages (28 to 82%) have been obtained for earthworms (32). In addition, the percentage of the total microbial cells in soils that are detected by fluorescence in situ hybridization with the bacterial probe EUB338 increases 3-to 12-fold during passage through the gut of L. terrestris, presumably because the ribosome contents of the bacteria increase after activation in the gut (16). Production of intestinal mucus by the earthworm may be an important factor for such activation, and it has been proposed that the digestive system of the earthworm is mutualistic (42).…”

Section: Discussionsupporting

confidence: 77%

“…Indeed, the total cell counts in the gut of the earthworm Lumbricus terrestris are only one-to sevenfold higher than the total cell counts in the surrounding soil (36). After comparing the numbers obtained with the cultivation methods used in the present study with the total cell counts in the earthworm gut and soil (16,36), one can speculate that the microorganisms cultured from the earthworm gut accounted for 1 to 6% of the total microorganisms in the gut, whereas the microorganisms cultured from the soil accounted for only 0.1% of the total microorganisms in the soil. While similar numbers have been reported for soils (2), even higher percentages (28 to 82%) have been obtained for earthworms (32).…”

Section: Discussionmentioning

confidence: 65%

N ₂ O-Producing Microorganisms in the Gut of the Earthworm Aporrectodea caliginosa Are Indicative of Ingested Soil Bacteria

Ihssen

Horn

Matthies

et al. 2003

The main objectives of this study were (i) to determine if gut wall-associated microorganisms are responsible for the capacity of earthworms to emit nitrous oxide (N 2 O) and (ii) to characterize the N 2 O-producing bacteria of the earthworm gut. The production of N 2 O in the gut of garden soil earthworms (Aporrectodea caliginosa) was mostly associated with the gut contents rather than the gut wall. Under anoxic conditions, nitrite and N 2 O were transient products when supplemental nitrate was reduced to N 2 by gut content homogenates. In contrast, nitrite and N 2 O were essentially not produced by nitrate-supplemented soil homogenates. The most probable numbers of fermentative anaerobes and microbes that used nitrate as a terminal electron acceptor were approximately 2 orders of magnitude higher in the earthworm gut than in the soil from which the earthworms originated. The fermentative anaerobes in the gut and soil displayed similar physiological functionalities. A total of 136 N 2 O-producing isolates that reduced either nitrate or nitrite were obtained from high serial dilutions of gut homogenates. Of the 25 representative N 2 O-producing isolates that were chosen for characterization, 22 isolates exhibited >99% 16S rRNA gene sequence similarity with their closest cultured relatives, which in most cases was a soil bacterium, most isolates were affiliated with the gamma subclass of the class Proteobacteria or with the gram-positive bacteria with low DNA G؉C contents, and 5 isolates were denitrifiers and reduced nitrate to N 2 O or N 2 . The initial N 2 O production rates of denitrifiers were 1 to 2 orders of magnitude greater than those of the nondenitrifying isolates. However, most nondenitrifying nitrate dissimilators produced nitrite and might therefore indirectly stimulate the production of N 2 O via nitrite-utilizing denitrifiers in the gut. The results of this study suggest that most of the N 2 O emitted by earthworms is due to the activation of ingested denitrifiers and other nitrate-dissimilating bacteria in the gut lumen.

“…The technological advances that allow the identification and monitoring of uncultivated (or uncultivable) bacteria have provided the means with which to reexamine this and other, similar, eukaryotic-eubacterial associations (2,12,16,18,21,47). Our work on microbial symbioses in the genus Prionitis has furnished a biological context within which to view SSU rDNA sequence differences among these gall-forming microbes.…”

Section: Discussionmentioning

confidence: 99%

Molecular and Ecological Evidence for Species Specificity and Coevolution in a Group of Marine Algal-Bacterial Symbioses

Ashen

Goff

2000

102

The phylogenetic relationships of bacterial symbionts from three gall-bearing species in the marine red algal genus Prionitis (Rhodophyta) were inferred from 16S rDNA sequence analysis and compared to host phylogeny also inferred from sequence comparisons (nuclear ribosomal internal-transcribed-spacer region). Gall formation has been described previously on two species of Prionitis, P. lanceolata (from central California) and P. decipiens (from Peru). This investigation reports gall formation on a third related host, Prionitis filiformis. Phylogenetic analyses based on sequence comparisons place the bacteria as a single lineage within the Roseobacter grouping of the ␣ subclass of the division Proteobacteria (99.4 to 98.25% sequence identity among phylotypes). Comparison of symbiont and host molecular phylogenies confirms the presence of three gallbearing algal lineages and is consistent with the hypothesis that these red seaweeds and their bacterial symbionts are coevolving. The species specificity of these associations was investigated in nature by whole-cell hybridization of gall bacteria and in the laboratory by using cross-inoculation trials. Whole-cell in situ hybridization confirmed that a single bacterial symbiont phylotype is present in galls on each host. In laboratory trials, bacterial symbionts were incapable of inducing galls on alternate hosts (including two non-gall-bearing species). Symbiont-host specificity in Prionitis gall formation indicates an effective ecological separation between these closely related symbiont phylotypes and provides an example of a biological context in which to consider the organismic significance of 16S rDNA sequence variation.Marine bacteria are associated with gall formation (tumorigenesis) on a number of species of red algae, although only two reports detail the specific causation of an algal gall by an identified bacterium (5, 10). This probably reflects the general situation encountered when attempting to cultivate, or isolate in pure culture, symbiotic microbes (10,33,44). In the red algal genus Prionitis (Rhodophyta, Halymeniaceae, Gigartinales) gall formation is known from at least four species world wide and is, in the case of Prionitis lanceolata (from central California), associated with the presence of a specific microorganism (5). Despite attempts by several authors, this bacterium has yet to be cultivated or isolated in pure culture (3)(4)(5). No physiological function of these bacterially induced Prionitis galls has been determined, nor is it apparent what, if any, benefit is derived by algal host or bacterial invader. This organismic relationship is termed a symbiosis sensu DeBary as used by Smith, meaning simply the living together of differently named organisms (43). The 16S rDNA phylotype of this eubacterium has been determined from its complete small-subunit ribosomal DNA sequence and whole-cell hybridization used to confirm the inductive role of this symbiont in gall formation (5).The purpose of this investigation was to determine if bacterial gall ...