Comparative transcriptomics of the nematode gut identifies global shifts in feeding mode and pathogen susceptibility
- James W. Lightfoot†1,
- Veeren M. Chauhan2,
- Jonathan W. Aylott2 and
- Christian Rödelsperger†1Email author
© Lightfoot et al. 2016
Received: 15 September 2015
Accepted: 25 January 2016
Published: 5 March 2016
The nematode Pristionchus pacificus has been established as a model for comparative studies using the well known Caenorhabditis elegans as a reference. Despite their relatedness, previous studies have revealed highly divergent development and a number of morphological differences including the lack of a pharyngal structure, the grinder, used to physically lyse the ingested bacteria in C. elegans.
To complement current knowledge about developmental and ecological differences with a better understanding of their feeding modes, we have sequenced the intestinal transcriptomes of both nematodes. In total, we found 464 intestine-enriched genes in P. pacificus and 724 in C. elegans, of which the majority (66 %) has been identified by previous studies. Interestingly, only 15 genes could be identified with shared intestinal enrichment in both species, of which three genes are Hedgehog signaling molecules supporting a highly conserved role of this pathway for intestinal development across all metazoa. At the level of gene families, we find similar divergent trends with only five families displaying significant intestinal enrichment in both species. We compared our data with transcriptomic responses to various pathogens. Strikingly, C. elegans intestine-enriched genes showed highly significant overlaps with pathogen response genes whereas this was not the case for P. pacificus, indicating shifts in pathogen susceptibility that might be explained by altered feeding modes.
Our study reveals first insights into the evolution of feeding systems and the associated changes in intestinal gene expression that might have facilitated nematodes of the P. pacificus lineage to colonize new environments. These findings deepen our understanding about how morphological and genomic diversity is created during the course of evolution.
Superficially, nematodes can be regarded as rather simple animals. They have a simple body plan, which in case of Caenorhabditis elegans is the outcome of a completely deterministic developmental proccess resulting in a fixed number of cells and large parts of their bodies are composed of two organs serving digestion and reproduction. However, the fact that nematodes form one of the most successful animal phyla and individual nematode species have invaded almost all ecological niches suggest that their relatively simple developmental program harbors enormous potential for adaptation to complex environments. This includes multiple independent events leading to the evolution of parasites that adapted to a diverse range of host environments (see [1, 2] for review). To understand, how such immense phenotypic and genotypic diversity is generated, is one of the key questions in evolutionary biology.
Over the last two decades, the nematode Pristionchus pacificus has been established as a satellite model organism to the widely known C. elegans for comparative studies involving developmental biology [3, 4], neuroscience [5, 6], immunity [7, 8], as well as comparative and population genomics [9, 10]. Despite the fact that both C. elegans and P. pacificus belong to the same taxonomic subgroup, Rhabditina, within nematodes , work on P. pacificus has revealed highly divergent patterns even involving newly acquired phenotypic traits  as well as novel genes . One of the most striking examples of a novel trait in P. pacificus is the presence of a mouthform plasticity in Pristionchus nematodes. This describes an environmentally controlled irreversible decision to develop either one mouthform that is better suited for bacterial feeding or another mouthform that allows predation on other nematodes . A second important morphological difference between C. elegans and P. pacificus nematodes, is the absence of a pharyngal structure in the terminal bulb of P. pacificus. The so called grinder is used to physically lyse bacteria in C. elegans. Therefore, typically, intact bacteria are not found in the gut of C. elegans, however, mutants defective in grinder formation exhibit intact bacteria in the gut . It has been shown that the grinder was lost in diplogastrid nematodes to which P. pacificus belongs  and it also has been suggested that the absence of the grinder has important consequences on the susceptibility to certain pathogens  potentially leading to a greater resistance in P. pacificus. Obviously, these rather dramatic morphological differences between P. pacificus and C. elegans likely reflect different lifestyles and environments. While Pristionchus nematodes are found in a necromenic association with scarab beetles , so far, the ecology of C. elegans is only recently beginning to be understood [16, 17]. However, based on population genetic analysis, a recent bottle neck and strong selective sweeps in the last centuries suggested that the dispersal of C. elegans might be linked to human migration patterns .
To complement current knowledge about developmental and ecological differences between both nematodes with a better understanding of the differences in feeding modes, we have sequenced the intestinal transcriptomes of C. elegans and P. pacificus. Using previously published data sets of intestine-enriched genes to assess the quality of our C. elegans intestinal transcriptome, we use the P. pacificus intestinal transcriptome to ask, to what extent are the intestinal transcriptomes conserved and whether transcriptomic differences have implications on the intestinal environment and on susceptibility to certain pathogens.
Dissection of nematode intestines and RNA extraction
Young adult C. elegans (N2) and P. pacificus (PS312) nematodes were selected from NGM plates seeded with Escherichia coli (OP50). Animals were picked into 20 \(\upmu\)l M9 on a glass slide and carefully decapitated using a fine needle. Intestines were gently extracted and cut from the carcass which was subsequently disposed of while the intestines were suspended separately in 50 \(\upmu\)l of M9 in an Eppendorf tube. In total 250 intestines from each species were collected and processed for RNA extraction. The intestinal RNA was purified using an Invitrogen PureLink RNA Micro Kit (Catalog no. 12183-016) with slight modifications. Briefly, the intestines were incubated for 5 minutes with 250 \(\upmu\)l TRIzol at room temperature before the addition of 70 \(\upmu\)l chloroform and a further 2–3 min incubation. The samples were then centrifuged at 13,000 rpm at 40 \(^\circ\)C for 15 min and the upper phase containing the RNA transferred to a new tube and an equal volume of 100 % ethanol added. The binding, wash and elution steps were performed as described in the manufacturers manual.
Transcriptome sequencing and analysis
RNA-seq libraries were generated using the Illumina TruSeq protocol and were sequenced as 100 bp paired ends in one multiplexed lane of an Illumina HiSeq 2000 resulting in 38,836,876 reads for the C. elegans intestine, 49,743,412 reads for the C. elegans whole animals, 47,369,694 reads for the P. pacificus intestine, and 42,912707 reads for the P. pacificus whole worms. Raw reads have been submitted to the NCBI short read archive under the study accessions: SRP061927 and SRP061928. We mapped raw reads to the C. elegans (WS230) and P. pacificus (Hybrid1) genome assemblies using tophat (version v2.0.3) and ran Cuffdiff (version v2.0.1) for differential gene expression analysis using the C. elegans (version WS230) gene annotations and the P. pacificus (version TAU) gene annotations. Protein domain annotations as well as orthology assignments were taken from [9, 19].
Imaging P. pacificus luminal pH with extended dynamic range pH sensitive nanosensors
Extended dynamic range pH-sensitive nanosensors were produced and calibrated as reported previously . Young adult P. pacificus nematodes were selected from synchronized NGM plates for intestinal pH measurements and immobilised on agarose pads. However, unlike in C. elegans, nanosensors were not maintained in the intestinal lumen of P. pacificus via feeding, therefore extended dynamic range pH-sensitive nanosensors (30 mg/ml) were introduced into the lumen of the P. pacificus intestine via microinjection. After successful injection, nematodes were placed onto OP50 seeded NGM plates and allowed to recover for 10 min before again being immobilised on fresh agarose pads for imaging. Green and red fluorescent channels were acquired on an Olympus FV 1000 confocal microscope and subsequently images were processed using MATLAB and FIJI open source software as previously described . The pixel-wise ratio of green and red fluorescent channels facilitated accurate real-time pH analyses which were subsequently displayed as a false colour pH heat map.
Identification of intestine-enriched genes in C. elegans and P. pacificus
Differential expression analysis identified 724 C. elegans and 464 P. pacificus genes that are enriched in the intestine as compared to whole animals (Additional file 1). The intestinal transcriptome of C. elegans has already been subject to several studies [23–26]. Pauli et al. immunopreciptated a poly-A tail binding protein that was transcribed from an intestine-specific promoter and identified 624 intestine enriched genes by comparison against muscle and germline specific expression . McGhee et al. hand-dissected the intestines from two thousand gonad-less C. elegans glp-4(bn2) animals and identified 80 intestine-enriched genes by comparison against transcriptome data obtained from intact adults . Spencer et al. used a cell sorting approach to isolate GFP labeled cells of various tissues followed by gene expression profiling on tiling arrays. Based on a comparison to whole worm expression data, they identified 924 intestine-enriched genes from late C. elegans embryos . Haenni et al. employed a protocol to isolate GFP-labeled intestinal nuclei and compared the transcriptome of intestinal nuclei to a transcriptome from unsorted nuclei, resulting in a candidate gene set of 2456 intestinal-enriched genes . We compared our set of 724 intestine-enriched genes with all four previous C. elegans intestinal transcriptome profiling studies (Fig. 1c). Despite drastic differences in previous approaches (various protocols to enrich for intestinal transcripts, different developmental stages, usage of mutant lines), which might explain to a large extent discrepancies in the shared gene sets (Fig. 1c), 477 (66 %) of our C. elegans intestine-enriched genes were identified previously by at least one other study. Thus, our data set is in good agreement with previous studies of C.elegans intestinal transcriptomes [23–26]. To investigate similarities across different gene sets at a different level, we repeated gene ontology (GO) enrichment analysis for all five different C. elegans data sets. Interestingly, not a single GO term was significantly enriched in all five data sets (Fig. 1d). The most robustly identified GO terms are all related to fatty acid metabolism. All of these most frequently found GO terms were also found based on our data, again supporting that our study is to a large extent in agreement with common trends identified by previous studies [23, 25, 26].
Highly diverged intestinal transcriptomes between C. elegans and P. pacificus
C. elegans genes with P. pacificus one-to-one ortholog, which showed intestine -enriched expression in both nematodes
P. pacificus ortholog
Hedgehog receptor protein
Hedgehog receptor protein
Metal ion transporters
bZip transcription factor
Intestinal transcriptomes are dominated by different gene families
Intestinal luminal pH is maintained despite transcriptomic divergence
Transcriptomic divergence is reflected in differential response to pathogens
Intestine-enriched genes in both species were tested for overlap with genes differentially expressed upon pathogen exposure
B. thuringiensis/ down
B. thuringiensis/ up
S. aureus/ down
S. aureus/ up
S. marcescens/ down
S. marcescens/ up
X. nematophila/ down
X. nematophila/ up
B. thuringiensis/ down
B. thuringiensis/ up
S. aureus/ down
S. aureus/ up
S. marcescens/ down
S. marcescens/ up
X. nematophila/ down
X. nematophila/ up
Strikingly, while none of the comparisons for P. pacificus showed a highly significant enrichment of intestine-enriched genes among genes differentially expressed upon pathogen exposure (P < 0.01), six out of eight comparisons showed highly significant associations between pathogen response and intestine-enriched genes. The only two exceptions consisted in comparisons with the two pathogens that killed C. elegans nematodes most efficiently, thus these transcriptomes are likely dominated by secondary effects such as pathogenesis related necrosis of host-tissues. In summary, our analysis clearly shows that intestine-enriched genes are associated with pathogen response in C. elegans but not in P. pacificus, which indicates that morphological differences in their feeding structures are paralleled by differences in pathogen susceptibilty.
In this study, we have investigated the intestinal transcriptomes of the nematodes C. elegans and P. pacificus. Our approach used RNA obtained from intact animals as control to screen for genes that are preferentially expressed in the intestinal sample. However, failure to detect the expression of a gene in the intestine, does not imply that the gene does not play a functionally important role in the intestine. Thus, many genes may be missed just because their overall expression level is not significantly different between the intestine and the complete animal or alternatively because of low statistical power (low expression, only one replicate). Our analysis showed that at least for a subset of genes, lack of conserved intestine-specific expression is due to broad expression in the other lineage (Fig. 2b, c). Thus, the identified gene sets provides just a footprint of the strongest intestine-specific expression and can be used for a first comparative analysis but they do not represent a complete catalogue of intestine-enriched genes. The fact, that previous studies have used quite distinct approaches to obtain tissue-specific transcriptomes in C. elegans [23–26], indicates that these kind of studies are inherently difficult and upscaling to more tissues and replicates only becomes feasible if sample and library preparation protocols further improve.
Despite the fact, that the identified gene sets are far from being complete, our first analysis shows substantial divergence between the genes with strongest intestine-enriched expression in both species. More precisely, only 15 genes with one-to-one orthologs were identified as intestine-enriched in both species (Fig. 2a), indicating that the largest fraction of intestine-enriched genes derived from lineage-specific events. Similarly, the strongest overrepresentations of gene families among intestine-enriched genes also seems to be lineage-specific (Fig. 3c, d), a pattern that recapitulates findings from studying the developmental transcriptomes of C. elegans and P. pacificus [3, 19]. Nevertheless, our analysis reveals highly conserved expression of certain Hedgehog signaling genes, which together with findings from other animal phyla [29, 30] points to an ancient and highly conserved function of Hedgehog signaling across all metazoans.
Given, that C. elegans and P. pacificus show strong morphological differences in their pharyngeal anatomy, i.e. the absence of the grinder in the P. pacificus lineage, which has been hypothesized to play a role in the susceptability to various pathogens , we compared the sets of intestine-enriched genes to genes that are differentially expressed upon pathogen exposure. Unexpectedly, we see a very striking trend as for C. elegans, there are highly significant overlaps between gene sets, while for P. pacificus there is not a single highly significant overlap (\(P<0.01\)). It has been shown that P. pacificus is more resistant to several pathogens compared to C. elegans  and it can be speculated that this is at least partially due to the sudden release of bacterial toxin upon physical lysis in C. elegans. Our data is largely consistent with this hypothesis and supports the idea of the lack of grinder as a mechanistic explanation for the increased resistance. However, the correlations that we see do not represent an experimental proof.
It has to be mentioned that the grinder serves to disrupt bacterial cell walls and to gain nutritients but it also serves as physical barrier to kill pathogenic bacteria and to prevent them from establishing intestinal infections . However, at least for intestinal pathogens such as S. marcescens, it has been shown that intestinal infections are facilitated by first interferring with the function of the grinder . More precisely, while normally, intact GFP labeled E. coli OP50 bacteria could not be observed in the gut of C. elegans worms, short exposure to a strain of S. marcescens enables fluorescent OP50 to pass the grinder . In addition, pathogenicity mechanisms can be very different even within a single pathogen. Pseudomonas aeruginosa for example has two different modes of killing C. elegans. A slow killing mode that functions via an infection-like proccess in the intestine and a fast toxin-based killing mode . Our interpretation of the lack of the grinder as a means to avoid high concentrations of bacterial toxins might therefore be better suited to explain the increased resistance to toxin-based pathogenicity mechanisms.
Taken together, The susceptibility to pathogens might rather be a question of being able to maintain the microbiome composition at correct concentrations throughout the intestine and both species might have developed different control mechanisms given their anatomy. Thus, any perturbation may cause a suboptimal state leading to an increased susceptibility. This is shown by the fact that grinder-less mutants or mutations affecting intestinal peristalsis show increased susceptibility at least to certain pathogens . In adition, to better understand the differences in pathogen suceptibility between both nematodes, we have to know how long bacteria stay resident in both species. Although comparisons of reported pumping rates and defectation cycles suggest differences between the species [8, 36, 37], a comprehensive analysis of bacterial residence times in both species is still lacking.
Our study reveals first insights into the evolution of feeding systems and the associated changes in intestinal gene expression and it provides support for the idea that anatomical differences might have facilitated nematodes of the P. pacificus lineage to colonize new habitats such as decaying beetle carcasses. These findings deepen our understanding about how morphological and genomic diversity is created during the course of evolution.
JWL conceived the study and carried out the expression profiling experiments. CR analysed the transcriptomic data. VMC and JWA did the intestinal pH measurements and corresponding data analysis. JWL and CR wrote the manuscript. All authors read and approved the final manuscript.
We would like to thank two anonymous reviewers for very helpful comments.
Availability of data
Raw reads have been submitted to the NCBI short read archive under the study accessions: SRP061927 and SRP061928. Genome and annotations are available at http://www.pristionchus.org
The authors declare that they have no competing interests.
This study does not involve research on humans or human material and also not on animals according to the german animal protection legislation. Therefore no ethical approval is needed.
This work was funded by the Max Planck Society.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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