Culture-independent investigation of the microbiome associated with the nematode Acrobeloides maximus

Abstract

Background: Symbioses between metazoans and microbes are widespread and vital to many ecosystems. Recent work with several nematode species has suggested that strong associations with microbial symbionts may also be common among members of this phylu. In this work we explore possible symbiosis between bacteria and the free living soil bacteriovorous nematode Acrobeloides maximus.

Methodology: We used a soil microcosm approach to expose A. maximus populations grown monoxenically on RFP labeled Escherichia coli in a soil slurry. Worms were recovered by density gradient separation and examined using both culture-independent and isolation methods. A 16S rRNA gene survey of the worm-associated bacteria was compared to the soil and to a similar analysis using Caenorhabditis elegans N2. Recovered A. maximus populations were maintained on cholesterol agar and sampled to examine the population dynamics of the microbiome.

Results: A consistent core microbiome was extracted from A. maximus that differed from those in the bulk soil or the C. elegans associated set. Three genera, Ochrobactrum, Pedobacter, and Chitinophaga, were identified at high levels only in the A. maximus populations, which were less diverse than the assemblage associated with C. elegans. Putative symbiont populations were maintained for at least 4 months post inoculation, although the levels decreased as the culture aged. Fluorescence in situ hybridization (FISH) using probes specific for Ochrobactrum and Pedobacter stained bacterial cells in formaldehyde fixed nematode guts.

Conclusions: Three microorganisms were repeatedly observed in association with Acrobeloides maximus when recovered from soil microcosms. We isolated several Ochrobactrum sp. and Pedobacter sp., and demonstrated that they inhabit the nematode gut by FISH. Although their role in A. maximus is not resolved, we propose possible mutualistic roles for these bacteria in protection of the host against pathogens and facilitating enzymatic digestion of other ingested bacteria.

Figure 1. A consistent bacterial population is associated with soil exposed A. maximus nematodes.

Samples isolated from A. maximus (N = 72, 93, 92 for Acro1-3) are dominated by Sphingobacteria and α-Proteobacteria after 24 h soil exposure. The bacteria from the identically treated soil sample (N = 94) contain a much higher percentage of Bacilli, and those associated with C. elegans after 24 h soil exposure (N = 94) are more broadly distributed across taxa.

Figure 2. Within the dominant subgroups the Acrobeloides maximus populations were consistent and limited compared to C.

elegans associated bacteria. A) The alpha-proteobacteria within A. maximus were predominantly Ochrobactrum. B) The Sphingobacteria within A. maximus were predominantly Pedobacter, with a consistent representation of Chitinophaga at a lower level. The C. elegans samples were more diverse at the genus level (both panels).

Figure 3. Long term culture experiments sampled at 3 wks, 2 m, 4 m, and 6 m post inoculation appear to show stability at the phylum level (A) but reveal shifts in population as the worm population ages at the genus level (B,C).

At 6 m post exposure the initially dominant clades have been supplanted in both the Sphingobacteria (B) and Alpha-Proteobacteria (C).

Figure 4. Phylogenetic tree created using Bayesian inference on the Ochrobactrum 16S rDNA sequences obtained from sample Acro2, the isolated Ochrobactrum sp., and several related sequences from the literature (see Materials and Methods).

Rhizobium leguminosarum bv. trifolii WSM597 was used as the outgroup for this analysis.

Figure 5. Phylogenetic tree created using Bayesian inference on the Pedobacter 16S rDNA sequences obtained from sample Acro2, the isolated Pedobacter sp., and several related sequences from the literature (see Materials and Methods).

Clone A (Heterodera) and Clone B (Heterodera) are partial sequences from Nour et al . Chitinophaga terrae was used as the outgroup for this analysis.

Figure 6. Phylogenetic tree created using Bayesian inference on the Chitinophaga 16S rDNA sequences obtained from sample Acro2, and several related sequences from the literature (see Materials and Methods).

Clone A Heterodera and Clone B Heterodera were previously identified microbial sequence tags recovered from the Soybean Cyst Nematode . Flavobacterium johnsoniae UW101 was used as the outgroup for this analysis.

Figure 7. Fluorescence in situ hybridization to identify Ochrobactrum (green) and Pedobacter (red) in the gut of formaldehyde fixed A. maximus after recovery from soil microcosm.

Samples from soil microcosms (A–D) and monoxenic culture on E. coli DH5μ (E, F) were imaged by DIC (A,C,E) or epifluorescence (B,D,F). Scale bar = 10 microns.