Bursaphelenchus xylophilus is the nematode responsible for a devastating epidemic of pine wilt disease in Asia and Europe, and represents a recent, independent origin of plant parasitism in nematodes, ecologically and taxonomically distinct from other nematodes for which genomic data is available. As well as being an important pathogen, the B. xylophilus genome thus provides a unique opportunity to study the evolution and mechanism of plant parasitism. Here, we present a high-quality draft genome sequence from an inbred line of B. xylophilus, and use this to investigate the biological basis of its complex ecology which combines fungal feeding, plant parasitic and insect-associated stages. We focus particularly on putative parasitism genes as well as those linked to other key biological processes and demonstrate that B. xylophilus is well endowed with RNA interference effectors, peptidergic neurotransmitters (including the first description of ins genes in a parasite) stress response and developmental genes and has a contracted set of chemosensory receptors. B. xylophilus has the largest number of digestive proteases known for any nematode and displays expanded families of lysosome pathway genes, ABC transporters and cytochrome P450 pathway genes. This expansion in digestive and detoxification proteins may reflect the unusual diversity in foods it exploits and environments it encounters during its life cycle. In addition, B. xylophilus possesses a unique complement of plant cell wall modifying proteins acquired by horizontal gene transfer, underscoring the impact of this process on the evolution of plant parasitism by nematodes. Together with the lack of proteins homologous to effectors from other plant parasitic nematodes, this confirms the distinctive molecular basis of plant parasitism in the Bursaphelenchus lineage. The genome sequence of B. xylophilus adds to the diversity of genomic data for nematodes, and will be an important resource in understanding the biology of this unusual parasite. Bursaphelenchus xylophilus is an important plant pathogen, responsible for an epidemic of pine wilt disease in Asia and Europe. B. xylophilus has acquired the ability to parasitise plants independently from other economically important nematodes and has a complex life cycle that includes fungal feeding and a stage associated with an insect, as well as plant parasitism. We have sequenced the genome of B. xylophilus and used it as a resource to understand disease mechanisms and the biological basis of its complex ecology. The ability to break down cellulose, the major component of the plant cell wall, is a major problem for plant parasitic nematodes as few animals can produce the required enzymes (cellulases). Previous work has shown that other plant parasitic nematodes have acquired cellulases from bacteria but we show that all Bursaphelenchus cellulases were most likely acquired independently from fungi. We also describe a complex set of genes encoding enzymes that can break down proteins and other molecules, perhaps reflecting the range of organisms with which B. xylophilus interacts during its life cycle. The genome sequence of Bursaphelenchus represents an important step forward in understanding its biology, and will contribute to efforts to control the devastating disease it causes.

While RNA interference (RNAi) has been deployed to facilitate gene function studies in diverse helminths, parasitic nematodes appear variably susceptible. To test if this is due to inter-species differences in RNAi effector complements, we performed a primary sequence similarity survey for orthologs of 77 Caenorhabditis elegans RNAi pathway proteins in 13 nematode species for which genomic or transcriptomic datasets were available, with all outputs subjected to domain-structure verification. Our dataset spanned transcriptomes of Ancylostoma caninum and Oesophagostomum dentatum, and genomes of Trichinella spiralis, Ascaris suum, Brugia malayi, Haemonchus contortus, Meloidogyne hapla, Meloidogyne incognita and Pristionchus pacificus, as well as the Caenorhabditis species C. brenneri, C. briggsae, C. japonica and C. remanei, and revealed that: (i) Most of the C. elegans proteins responsible for uptake and spread of exogenously applied double stranded (ds)RNA are absent from parasitic species, including RNAi-competent plant-nematodes; (ii) The Argonautes (AGOs) responsible for gene expression regulation in C. elegans are broadly conserved, unlike those recruited during the induction of RNAi by exogenous dsRNA; (iii) Secondary Argonautes (SAGOs) are poorly conserved, and the nuclear AGO NRDE-3 was not identified in any parasite; (iv) All five Caenorhabditis spp. possess an expanded RNAi effector repertoire relative to the parasitic nematodes, consistent with the propensity for gene loss in nematode parasites; (v) In spite of the quantitative differences in RNAi effector complements across nematode species, all displayed qualitatively similar coverage of functional protein groups. In summary, we could not identify RNAi effector deficiencies that associate with reduced susceptibility in parasitic nematodes. Indeed, similarities in the RNAi effector complements of RNAi refractory and competent nematode parasites support the broad applicability of this research genetic tool in nematodes. Many organisms regulate gene expression through an RNA interference (RNAi) pathway, first characterized in the nematode Caenorhabditis elegans. This pathway can be triggered experimentally using double-stranded (ds)RNA to selected gene targets, thereby allowing researchers to ‘silence’ individual genes and so investigate their function. It is hoped that this technology will facilitate gene silencing in important parasitic nematodes that impose a considerable health and economic burden on mankind. Unfortunately, differences in RNAi susceptibility have been observed between species. Here we investigated the possibility that differences in the complement of effector proteins involved in the RNAi pathway are responsible for these differences in susceptibility. Our data revealed that most facets of the RNAi pathway are well represented across parasitic nematodes, although there were fewer pathway proteins in other nematodes compared to C. elegans. In contrast, the proteins responsible for uptake and spread of dsRNA are not well represented in parasitic nematodes. However, the importance of these differences is undermined by our observation that the protein complements in all the parasites were qualitatively similar, regardless of RNAi-susceptibility. Clearly, differences in the RNAi pathway of parasitic nematodes do not explain the variations in susceptibility to experimental RNAi.

Plant parasitic nematodes (PPNs) seriously threaten global food security. Conventionally an integrated approach to PPN management has relied heavily on carbamate, organophosphate and fumigant nematicides which are now being withdrawn over environmental health and safety concerns. This progressive withdrawal has left a significant shortcoming in our ability to manage these economically important parasites, and highlights the need for novel and robust control methods. Nematodes can assimilate exogenous peptides through retrograde transport along the chemosensory amphid neurons. Peptides can accumulate within cells of the central nerve ring and can elicit physiological effects when released to interact with receptors on adjoining cells. We have profiled bioactive neuropeptides from the neuropeptide-like protein (NLP) family of PPNs as novel nematicides, and have identified numerous discrete NLPs that negatively impact chemosensation, host invasion and stylet thrusting of the root knot nematode Meloidogyne incognita and the potato cyst nematode Globodera pallida. Transgenic secretion of these peptides from the rhizobacterium, Bacillus subtilis, and the terrestrial microalgae Chlamydomonas reinhardtii reduce tomato infection levels by up to 90% when compared with controls. These data pave the way for the exploitation of nematode neuropeptides as a novel class of plant protective nematicide, using novel non-food transgenic delivery systems which could be deployed on farmer-preferred cultivars. Plant parasitic nematodes (PPN) reduce crop plant yield globally, undermining food security. Many of the chemicals used to kill these parasites are non-specific and highly toxic, and are being phased out of general use through governmental and EU regulation. The withdrawal of these chemicals is beneficial to the environment, but limits our ability to protect crops from infection. Efforts must now focus on developing environmentally safe PPN controls. PPNs can absorb various molecules directly from the environment into their nervous system, including peptides and proteins. Here we profiled the feasibility of using PPN neuropeptides, small signalling molecules, to interfere with normal PPN behaviour. We exposed PPNs to a variety of neuropeptides, and found that they could interfere with behaviours that are important to host-finding and invasion. We then developed soil-dwelling microbes that could generate and secrete these neuropeptides into the soil where the PPN infective juveniles are found. These transgenic microbes can protect host plants from infection, and represent a completely new approach to controlling PPNs in crop plants. Importantly, these neuropeptides appear to have no impact on other beneficial nematodes found in the soil.

The rationale for identifying drug targets within helminth neuromuscular signalling systems is based on the premise that adequate nerve and muscle function is essential for many of the key behavioural determinants of helminth parasitism, including sensory perception/host location, invasion, locomotion/orientation, attachment, feeding and reproduction. This premise is validated by the tendency of current anthelmintics to act on classical neurotransmitter-gated ion channels present on helminth nerve and/or muscle, yielding therapeutic endpoints associated with paralysis and/or death. Supplementary to classical neurotransmitters, helminth nervous systems are peptide-rich and encompass associated biosynthetic and signal transduction components - putative drug targets that remain to be exploited by anthelmintic chemotherapy. At this time, no neuropeptide system-targeting lead compounds have been reported, and given that our basic knowledge of neuropeptide biology in parasitic helminths remains inadequate, the short-term prospects for such drugs remain poor. Here, we review current knowledge of neuropeptide signalling in Nematoda and Platyhelminthes, and highlight a suite of 19 protein families that yield deleterious phenotypes in helminth reverse genetics screens. We suggest that orthologues of some of these peptidergic signalling components represent appealing therapeutic targets in parasitic helminths.