Culture-Independent Investigation of the Microbiome Associated with the Nematode <i>Acrobeloides maximus</i>

<div><p>Background</p><p>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 <i>Acrobeloides maximus</i>.</p><p>Methodology</p><p>We used a soil microcosm approach to expose <i>A. maximus</i> populations grown monoxenically on RFP labeled <i>Escherichia coli</i> 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 <i>Caenorhabditis elegans</i> N2. Recovered <i>A. maximus</i> populations were maintained on cholesterol agar and sampled to examine the population dynamics of the microbiome.</p><p>Results</p><p>A consistent core microbiome was extracted from <i>A. maximus</i> that differed from those in the bulk soil or the <i>C. elegans</i> associated set. Three genera, <i>Ochrobactrum</i>, <i>Pedobacter</i>, and <i>Chitinophaga</i>, were identified at high levels only in the <i>A. maximus</i> populations, which were less diverse than the assemblage associated with <i>C. elegans</i>. Putative symbiont populations were maintained for at least 4 months post inoculation, although the levels decreased as the culture aged. Fluorescence <i>in situ</i> hybridization (FISH) using probes specific for <i>Ochrobactrum</i> and <i>Pedobacter</i> stained bacterial cells in formaldehyde fixed nematode guts.</p><p>Conclusions</p><p>Three microorganisms were repeatedly observed in association with <i>Acrobeloides maximus</i> when recovered from soil microcosms. We isolated several <i>Ochrobactrum sp.</i> and <i>Pedobacter sp.</i>, and demonstrated that they inhabit the nematode gut by FISH. Although their role in <i>A. maximus</i> 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.</p></div

Identified Isolates from <i>A. maximus</i> microcosm and long term culture.

a<p>OTU assigned using RDP phylogeny. Isolates assigned to the same OTU were verified to be distinct using direct sequence comparison (bl2seq).</p

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

<p><i>Rhizobium leguminosarum</i> bv. trifolii WSM597 was used as the outgroup for this analysis.</p

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

<p>Clone A (Heterodera) and Clone B (Heterodera) are partial sequences from Nour et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067425#pone.0067425-Nour1" target="_blank">[21]</a>. <i>Chitinophaga terrae</i> was used as the outgroup for this analysis.</p

Within the dominant subgroups the <i>Acrobeloides maximus</i> populations were consistent and limited compared to <i>C.</i>

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

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

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

Shannon-Wiener Population Statistics for Microcosm Samples.

<p>Shannon-Wiener Population Statistics for Microcosm Samples.</p

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

<p>Clone A Heterodera and Clone B Heterodera were previously identified microbial sequence tags recovered from the Soybean Cyst Nematode <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067425#pone.0067425-Nour1" target="_blank">[21]</a>. <i>Flavobacterium johnsoniae</i> UW101 was used as the outgroup for this analysis.</p

A consistent bacterial population is associated with soil exposed <i>A.</i><i>maximus</i> nematodes.

<p>Samples isolated from <i>A. maximus</i> (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 <i>C. elegans</i> after 24 h soil exposure (N = 94) are more broadly distributed across taxa.</p

Analysis of surface behaviors to determine efficacy of Varpa_4680 (<i>wbu</i>B) and Varpa_5900 (<i>pil</i>Y1) complementation.

<p>Biofilm levels after 24 h incubation and swarm diameters at 48 h for wt and mutant strains expressing either <i>wbu</i>B (A, B) or <i>pil</i>Y1 (C, D) <i>in trans</i> compared to vector controls. 86 = Varpa_4680::Tn5, 223 = Varpa_5900::Tn5. Error was computed as +/−SEM. All p-values were calculated using the student's unpaired T-test. For all panels a = p<0.01 compared to wt+vec, b = p<0.01 compared to 86+vec (A,B) or 223+vec (C,D).</p