Infection by plant-parasitic nematodes (PPN) reduces global agricultural output by 10-15%. For rice alone this loss represents a commercial value of $35bn. All crops are affected to some degree, and the impact on subsistence farmers can be especially dire. The most pernicious PPN are highly adapted for life within plant roots. Some, such as the cyst (Globodera and Heterodera spp.) and root-knot nematodes (RKN: Meloidogyne spp.) establish a permanent feeding site by usurping host developmental circuitry. Others, including the lesion nematode (Pratylenchus spp.) function essentially as migratory herbivores. Understanding the molecular basis underpinning these distinct plant-nematode interactions is a key objective of the PPN research community.

The development of genetic tools for helminths has proven a challenge, largely because of the obligate, parasitic life stages within the host. For ethical and technical reasons, such barriers are much lower for PPN. Thus, we exploited M. hapla’s small diploid genome, plus its ability to both self- and out-cross, to develop necessary genetic resources. Parental lines (VW9 and LM) were established from field isolates that exhibited broad phenotypic diversity, and have been inbred and crossed. The resultant mapping populations (~100 RILs) segregate genotypic diversity, which we are correlating with nematode and plant phenotypes. One strategy has been a cross-species, eQTL analysis that samples each nematode gene to ask how it influences the expression of each plant gene. In parallel, each of the parents has been sequenced, and the VW9 transcriptome is now defined by more than a billion mapped Illumina reads derived from the RILs. The scaffolds from genome sequencing have been anchored to the 16 linkage groups of the map, and ~28,000 SNPs identified between the parents of our mapping populations. As more PPN genomes are obtained, it is clear that genome size and gene content can vary greatly within and between genera. For example, the M. hapla genome spans 54 Mb, whereas the genome of M. incognita is expanded to ~150 Mb. Recent analysis points to this species being a recent, interspecific hybrid, with a decaying, allo-hexaploid genome. At the other end of the spectrum, the 19.6 Mb Pratylenchus coffeae genome encodes a mere 6,700 genes, compared to ~14,500 for M. hapla. KEGG analysis revealed no obvious loss of biochemical ability as a result of being an obligate parasite. Broad comparisons, such as between P. coffeae, M. hapla, and C. elegans reveal patterns of gene family expansion/contraction, whereas closer comparisons (e.g., M. incognita vs M. hapla) provide a platform to understand evolution of host range, virulence and asexual reproduction.

However, despite the “taming” of M. hapla, several crucial deficiencies remain: 1) Seemingly trivial, cryopreservation is unreliable and largely ineffective. Currently, worm stocks must be continuously maintained on live plants; 2) Transformation remains elusive. In particular, gonadal microinjection is yet to be successful for RKN; 3) Mutagenesis is not practical. Unlike C. elegans, which can reproduce with huge loss of functionality, obligate parasites must complete complex interactions with their host in order to reproduce. Hence, in an obligate parasite, most genes are essential; 4) There is no lineage information for any PPN, limiting the scope of ablation experiments. Similarly, the anatomy is not well documented at cellular resolution. For example, it remains a debated point as to whether adult RKN retain a gut. Similarly, the mechanism of presumed muscle “degeneration” associated with the nematodes becoming sedentary is undocumented, as is the re-establishment of locomotion in males. Understanding how much anatomical and regulatory “equivalence” exists between C. elegans and PPN might be a useful guide. For example, is the logic of transcriptional regulation common to C. elegans and PPN (e.g., will M. hapla promoters drive GFP in an anticipated spatio-temporal pattern in C. elegans)? Can the 5 pharyngeal glands in C. elegans inform about the 3 such glands in PPN?

Although microbes influence the health and disease of all animals, we still understand little about mechanisms underlying microbial specificity for hosts and tissues or microbial contributions to host biology. To address these questions, my lab investigates a relatively simple tripartite association in which nematodes of the genus Steinernema parasitize insects by virtue of their mutualistic association with Xenorhabdus bacteria. The Steinernema-Xenorhabdus-insect symbiosis offers numerous advantages as a model to understand the specificity and processes of beneficial and pathogenic microbial colonization of host tissues. These include its experimental tractability: Each member of the tripartite association can be reared separately or together in varied combinations in the laboratory, at least one representative of each has a sequenced genome and transcriptome, and numerous genetic techniques have been developed for Xenorhabdus bacteria. Furthermore, investigations are simplified by the fact that Steinernema are colonized by a monoxenic culture of a specific species of Xenorhabdus bacteria, rather than a complex microbial consortium. Thus, the system can be probed to understand the molecular basis for specificity and the impacts of bacteria on animal host behavior, reproduction, immunity, and physiology. Finally, the Steinernema-Xenorhabdus mutualism is a conserved characteristic of both genera, facilitating evolutionary and comparative analyses of symbiosis mechanisms. The potential of the Steinernema-Xenorhabdus symbiosis to reveal fundamental molecular and cellular processes mediating symbiosis is inhibited by a lack of genetic and molecular tools to manipulate the nematode. Furthermore, although several C. elegans researchers have made advances in understanding several aspects of Steinernema molecular biology, much remains to be learned regarding Steinernema physiology, behavior, development, and immunity with respect to their symbiosis with Xenorhabdus.

Research in my lab focuses on understanding the molecular basis of bacterial symbiosis with animal hosts, and what distinguishes beneficial and pathogenic interactions. I will focus my talk on the molecular basis of species specificity between nematodes and bacteria. We have determined that X. nematophila bacterial association with its S. carpocapsae nematode host is tissue- and species-specific and requires the integral outer membrane protein NilB; Bacteria adhere to the anterior intestinal epithelial surface of adult and juvenile nematodes only when expressing nilB. The consequences of this surface colonization are as yet unknown, but possibilities include bacteria-host cell signaling that promotes pheromone production or immunity. The molecular function of NilB also is unknown. It is a beta-barrel protein with surface exposed loops both necessary for nematode interactions and a predicted small channel with an electronegative strip. These regions may be important for NilB binding to host cell surfaces or for transport of host-derived molecules. NilB is the only characterized member of a family of OMPs found exclusively in symbionts of animals (e.g., Neisseria, Haemophilus, Moraxella, Actinobacillus). Therefore, understanding NilB function in Xenorhabdus–Steinernema mutualism will provide insights into NilB homolog function in human associated microbes.

There are ~23,000 described species of nematodes, and an estimated 2-10 million species exist. Genomics research on nematodes (particularly Caenorhabditis elegans) has delivered important information on core biological processes. Adding additional nematode genomes to that of C. elegans will allow the specific instance of C. elegans to be contextualised, and will we hope feed research on comparative genomics of nematodes, the evolutionary biology of genome change, and the biology of the (many) parasitic nematodes, amongst other fields.

Why (only) 959 nematode genomes?

All adult female hermaphrodite C. elegans have 959 somatic cells. The definition of the embryonic lineage of C. elegans from fertilised zygote to fertile adult was a milestone in C. elegans developmental biology. Just as the tree of the C. elegans embryonic lineage was a key underpinning of later work on this model nematode, we hope that a Nematoda phylogeny with 959 genome-sequenced taxa will underpin the investigation of nematode biology in general. Next generation sequencing technologies already can deliver 100-fold coverage of a new genome in under a week: our challenge is to recruit the next generation of (bioinformatics-capable, genome-savvy) nematologists to drive this aspiration.

Schistosomiasis is a parasitic disease caused by blood flukes (Phylum Platyhelminthes) of the genus Schistosoma. As many as 800 million people are affected in parts of South America, the Western Pacific and China with the majority (>95%) living in sub-Saharan Africa.  Children as young as six months are infected with what can be a life-long affliction.  In the severest of cases, damage to internal organs can kill. However, the disease is better known for its insidious ability to rob those who already live at or below subsistence of their physical and cognitive vitality.  This translates to missed school days, decreased ability to work and lost potential regarding both personal and societal productivity. Often, schistosomiasis is just one of a number of overlapping parasitic diseases, including other worm infections, which when combined degrade the well-being of whole communities.

Although parasitic flatworms can display bewildering life-cycle complexity, schistosomes are relatively easy to keep in the lab requiring as hosts mice (or hamsters) and a robust fast-breeding snail vector – a fortunate situation given the importance of the global disease.  This ease has not only facilitated a wealth of ‘classical’ life-history, biochemical, host-pathogen, and drug discovery studies, but also advances in ‘-omic’ applications over the last ten years with continually-improving annotations of genome, transcriptome and proteome data supported by interrogable databases. In turn, there has been a real effort in and measured success with a range of ‘post-genomic’ reverse genetic tools, typically involving both transient RNAi and transgenesis, but also viral-mediated germ line transgenesis. These developments are timely given the growing concern in the schistosome community at the precarious reliance on just one drug, praziquantel, to treat ‘continents of people’ and the consequent need to find new drug and vaccine targets, and diagnostic candidates.  Nonetheless, a wish-list for a standardized schistosome ‘molecular tool kit’ would include such items as complete in vitro cultivation of the parasite without recourse to mammalian (or molluscan) hosts, targeted germ line integration and the generation of cell lines. On the flip side, an example of employing C. elegans as a functional screening tool for schistosome drug discovery is presented and this approach bodes well for future drug discovery efforts.  Finally, a list of resources available to those potentially interested in working with schistosomes, even ‘on the side,’ is provided.

In December 2012, the results of the much awaited Global Burden of Disease (GBD) Study 2010 were released.  Aside from visceral leishmaniasis, the most important neglected tropical diseases (NTDs) when measured in disability adjusted life years (DALYs) were helminth infections led by the intestinal nematode infections (with hookworm infection accounting for two-thirds of the DALYs lost) followed by schistosomiasis, lymphatic filariasis, and food-borne trematode infections.  Together these NTDs in addition to onchocerciasis and trachoma are being targeted for either “elimination” (LF, onchocerciasis, and trachoma) or “control” (the intestinal nematode infections and schistosomiasis) through a scaled up program of mass drug administration (MDA) with support from USAID, UKAID, and a private END (Ending Neglected Disease Fund), and under the auspices of a 2012 London Declaration and a 2013 World Health Assembly Resolution.  However, it is unclear whether these goals can be achieved without the development of additional or improved control tools, i.e., drugs, diagnostics, vaccines, and monitoring for resistance or other operational parameters.  For instance, failure with benzimidazole anthelminthics is widespread for hookworm and trichuriasis, especially when they are used in a single dose, although the basis of this observation is not well understood.  It is uncertain whether drug resistance has emerged and is an ongoing concern.  Moreover, there are fundamental aspects of the basic biology of intestinal nematodes that we do not understand such as tissue tropisms and ecological niches, and the basis of long-term survival both within and outside of the human host.   Similar questions remain also for schistosomes and food-borne trematodes (and for praziquantel Rx) and we are just beginning to understand the basis for helminth-induced carcinogenesis and schistosome-induced female urogenital schistosomiasis and HIV/AIDS transmission.  Early stage vaccines for hookwrom and schistosomiasis are also in clinical development.  There remain a number of outstanding questions on the fundamental biology of filarial worms that cause lymphatic filariasis, onchocerciasis, and loiasis, including the basis of parasite tropisms, longevity, and immune evasion, the basis of microfilarial periodicitiy, and how parasite death occurs and its link to host immunopathogenesis.  There is a need to develop a macrofilaricide but few leads on how to achieve drug or vaccine development for this purpose.  Finally there are several important human helminth infections, such as toxocariasis and strongyloidiasis, for which we know little about in terms of disease burden and yet which may be as important as the other helminthiases mentioned.  Human Strongyloides infection has a unique interface between pathogenesis and nematode developmental biology, while toxocariasis has emerged as an important helminth infection the United States and it many be linked to asthma, developmental delays, and epilepsy, especially among under represented minority populations.