The transcriptome of the invasive eel swimbladder nematode parasite Anguillicola crassus

Abstract

Background: Anguillicola crassus is an economically and ecologically important parasitic nematode of eels. The native range of A. crassus is in East Asia, where it infects Anguilla japonica, the Japanese eel. A. crassus was introduced into European eels, Anguilla anguilla, 30 years ago. The parasite is more pathogenic in its new host than in its native one, and is thought to threaten the endangered An. anguilla across its range. The molecular bases for the increased pathogenicity of the nematodes in their new hosts is not known.

Results: A reference transcriptome was assembled for A. crassus from Roche 454 pyrosequencing data. Raw reads (756,363 total) from nematodes from An. japonica and An. anguilla hosts were filtered for likely host contaminants and ribosomal RNAs. The remaining 353,055 reads were assembled into 11,372 contigs of a high confidence assembly (spanning 6.6 Mb) and an additional 21,153 singletons and contigs of a lower confidence assembly (spanning an additional 6.2 Mb). Roughly 55% of the high confidence assembly contigs were annotated with domain- or protein sequence similarity derived functional information. Sequences conserved only in nematodes, or unique to A. crassus were more likely to have secretory signal peptides. Thousands of high quality single nucleotide polymorphisms were identified, and coding polymorphism was correlated with differential expression between individual nematodes. Transcripts identified as being under positive selection were enriched in peptidases. Enzymes involved in energy metabolism were enriched in the set of genes differentially expressed between European and Asian A. crassus.

Conclusions: The reference transcriptome of A. crassus is of high quality, and will serve as a basis for future work on the invasion biology of this important parasite. The polymorphisms identified will provide a key tool set for analysis of population structure and identification of genes likely to be involved in increased pathogenicity in European eel hosts. The identification of peptidases under positive selection is a first step in this programme.

Figure 1

Annotation of the Anguilicolla crassus transcriptome. Number of annotated sequences in the transcriptome of A. crssus for all TUGs (a) and for highCA contigs (b). Annotations with Gene Ontology (GO), Enzyme Commission (EC) and Kyoto Encyclopedia of Genes and Genomes (KEGG) terms were inferred for predicted proteins using annot8r (version 1.1.1) [37]. For highCA contigs additional domain annotations obtained with InterProScan [39] are also enumerated.

Figure 2

Comparing high level GO-slim annotations. Comparing high level GO-slim annotations obtained through annot8r (version 1.1.1) [37] for A. crassus to those for the model nematodes C. elegans and B. malayi infered using the same pipeline. For GO categories molecular function, cellular compartment and biological process the number of terms in high level GO-slim categories is given. In the two parasitic nematodes a higher degree of congruence in annotation spectrum is observed (Spearman correlation coefficient 0.95) than in comarison to the complete proteome of C. elegans (0.90).

Figure 3

Enrichment of Signal-positives for categories of evolutionary conservations. Categories of evolutionary conservation recorded using the taxonomy of BLAST-matches at two different bitscore thresholds (50 or 80) are compared for the occurence of signal peptide cleavage sites and signal anchor signatures, predicted using SignalP V4.0 [38]. Contigs were categorised as conserved, novel in the kingdom Metazoa, the phylum Nematoda or Spirurina sensu[17]. TUGs without any match at a given threshold were categorised as novel in A. crassus (Ac). The highest proportions of genes predicted to have secretory signal peptides are observed in TUGs predicted to be part of gene families that arose in the last common ancestor of Nematoda or to be novel to A. crassus.