The phytohormone auxin plays a crucial role in the development of so-called syncytia induced by cyst nematodes upon feeding on plant roots. However, it is unknown whether nematode-derived auxin contributes to plant parasitism by cyst nematodes. To do so, plant-parasitic nematodes should be able to produce and release auxin into the environment. Here, we investigated whether this is the case. Mass-spectrometry analyses show that the cyst nematode Globodera pallida is able to synthesize and release the auxin indole-3-acetic acid (IAA) and indicates that its biosynthesis can be stimulated by root diffusate. Using a collection of known auxin biosynthesis genes from well-studied pathways in bacteria and plants we revealed that in the genome of G. pallida candidate genes are present for 5 major biosynthesis pathways, which could be responsible for auxin production. RNA-seq data supported the genomic analysis and revealed the upregulation of various candidate biosynthesis genes during parasitic life stages of the nematode, suggesting a possible role in plant parasitism. However, genomic analysis within the phylum Nematoda showed that the potential auxin biosynthesis genes are wide-spread and also occur in bacterivorous, fungivorous, entomopathogenic and animal-parasitic nematodes. Therefore, our data show that G. pallida is able to synthesize and release auxin into the environment, but that potential auxin biosynthesis pathways are not unique for plant-parasitic nematodes. So, auxin biosynthesis may play a broader role in the life history of nematodes. However, a more specialized role for nematode-produced auxin in plant parasitism cannot be excluded and needs further investigation.

The response of individual adult males of the potato cyst nematodes, Globodera rostochiensis and G. pallida, to sex pheromones from adult females was investigated using electrophysiological techniques. Each male nematode was pierced with an electrode close to the cephalic region and then exposed to pheromones from virgin females. Cellular responses in the form of action potentials were recorded as spike activity. The spike frequency produced by G. rostochiensis and G. pallida males increased significantly after the application of their homospecific pheromone. The spike frequency produced by G. pallida males also increased significantly after the application of G. rostochiensis female sex pheromone. In contrast, males of G. rostochiensis showed no significant response to G. pallida female sex pheromone. The electrophysiological results support and considerably extend information from agar plate behavioural bioassays.

A method is described for the isolation of high molecular weight DNA from females of potato cyst nematodes. Restriction enzyme analyses of repetitive DNA revealed 30 bands specific for Globodera rostochiensis and 8 bands specific for G. pallida. None of the repetitive DNA bands resolved was common to both species. Hybridization patterns, obtained with a DrosophilaβJ-tubulin probe, revealed 4 common bands, 3 bands specific for G. rostochiensis and 12 bands specific for G. pallida.

Sensitive assays capable of detecting proteinases in single females of the phytoparasite Globodera pallida have been developed and used to define the proteinase activity of young adult females. Digestion of the large subunit of the plant protein Rubisco established a pH optimum for the proteinase activity at pH 5·7. The activity was inhibited by the cysteine proteinase inhibitors p-chloromercuribenzoic acid (PMBA) and p-chloromercurisulphonic acid (PMSA) and stimulated by both cysteine and dithiothreitol (DTT). It was moderately reduced by L-trans-epoxysuccinyl-leucylamido-(4- guanidino) butane (E64) but not by specific inhibitors of serine, aspartate or metallo-proteinases. The activity separated into 3 bands on a non-denaturing gel but only I proteinase of 62 kDa was recovered following a combination of anion-exchange chromatography and affinity chromatography using PMBA. The effect of inhibitors was similar to that reported previously for some of the cysteine proteinase activity recovered from Caenorhabditis elegans but is apparently not that for which the corresponding gene has been cloned in this nematode and Haemonchus contortus. The proteinase may have a major role in digestion of dietary protein and so offers an exciting target for future control of this important plant-parasitic nematode.

A cDNA clone encoding a full length putative collagen has been isolated in a screen of a mixed stage Globodera pallida expression library. Comparison of the deduced amino acid sequence of this molecule with other collagens suggests it is a cuticular collagen and a member of the col-8 subfamily of collagen genes. Northern blots show the gene is expressed specifically in gravid, adult females of the parasite as compared to second (invasive) stage juveniles and virgin females. Preliminary immunocytochemical studies indicate this collagen is present in areas other than the cuticle; these findings and the potential functional role of this collagen are discussed.

Low doses of the acetylcholinesterase-inhibiting carbamate nematicides disrupt chemoreception in plant-parasitic nematodes. Fluorescein isothiocyanate (FITC)/dextran conjugates up to 12 kDa are taken up from the external medium by certain chemosensory neurons in Caenorhabditis elegans. Similar chemoreceptive neurons of the non-feeding infective stage of Heterodera glycines (soybean cyst nematode) fill with FITC and the nuclei of their cell bodies selectively stain with bisbenzimide. The widely used nematicide aldicarb disrupts the chemoreceptive response of H. glycines with 50% inhibition at very low concentrations (ca 1 pM), some 10−6-fold lower than required to affect locomotion. Similarly, the anthelmintic levamisole had this effect at 1 nM. Peptides selected as mimetics of aldicarb and levamisole also disrupt chemoreception in H. glycines and Globodera pallida at 10−3-fold or lower concentration than required to inhibit locomotion. We propose an uptake pathway for aldicarb, levamisole, peptide mimetics and other soluble molecules by retrograde transport along dendrites of chemoreceptive neurons to the cell bodies and synapses where they act. This may prove to be a general mechanism for the low-dose effects of some nematicides and anthelmintics.

Electrophoresis of secretions collected from Globodera pallida revealed a smeared region between 25 and 50 kDa, and a single band of <20 kDa. The secretions were used to raise an antiserum (LW1). Immunoblotting of parasite homogenates with LW1 differentiated G. pallida from its sibling species G. rostochiensis and revealed differences between different populations of G. pallida and G. rostochiensis. Indirect immunofluorescence studies with LW1 indicated that at least some of the secretions were surface localized and that antibody binding to the nematode surface was periodate sensitive. Periodate sensitivity indicated that these differences could be due to glycosylation differences. Glycosylation differences were also detected by blotting nematode homogenates with the lectin wheat germ agglutinin (WGA). WGA was also able to differentiate between G. rostochiensis which gave 2 bands at 130 kDa and 110 kDa, and G. pallida which produced 2 bands present at 120 kDa and 110 kDa. Further localization studies using immunoelectron microscopy demonstrated that antibody binding could be seen to secretions found in the pump chamber of the metacorpal bulb at the base of the stylet. From further specimens it could be observed that the contents of the subventral glands were heavily labelled, indicating that the material seen in the metacorpal bulb had originated from the subventral glands.