Getting to the root of neuronal signalling in plant-parasitic nematodes using RNA interference

A variety of genes expressed in preparasitic second-stage juveniles (J2) of plant-parasitic nematodes appear to be vulnerable to RNA interference (RNAi) in vitro by coupling double-stranded (ds)RNA soaking with the artificial stimulation of pharyngeal pumping. Also, there is mounting evidence that the in planta generation of nematode-specific double-stranded RNAs (dsRNAs) has real utility in the control of these pests. Although neuronally-expressed genes in Caenorhabditis elegans are commonly refractory to RNAi, we have discovered that neuronally-expressed genes in plant-parasitic nematodes are highly susceptible to RNAi and that silencing can be induced by simple soaking procedures without the need for pharyngeal stimulation. Since most front-line anthelmintics that are used for the control of nematode parasites of animals and humans act to disrupt neuromuscular coordination, we argue that intercellular signalling processes associated with neurons have much appeal as targets for transgenic plant-based control strategies for plant-parasitic nematodes. FMRFamide-like peptides (FLPs) are a large family of neuropeptides which are intimately associated with neuromuscular regulation, and our studies on flp gene function in plant-parasitic nematodes have revealed that their expression is central to coordinated locomotory activities. We propose that the high level of conservation in nervous systems across nematodes coupled with the RNAi-susceptibility of neuronally-expressed genes in plant-parasitic nematodes provides a valuable research tool which could be used to interrogate neuronal signalling processes in nematodes.

Novel bioassays to examine the host-finding ability of plant-parasitic nematodes

We present two novel bioassays to be used in the examination of plant-parasitic nematode host-finding ability. The host-finding 'pipette-bulb assay' was constructed from modelled Pasteur pipette bulbs and connecting barrels using parafilm fastenings. This assay examines the direction of second-stage juvenile (J2) migration in response to a host seedling, through a moistened sand substrate, which underlies terminal upward-facing 'seedling bulbs', one containing a host seedling in potting compost, the other with only potting compost. An equal watering regime through both upward-facing seedling bulbs creates a directional concentration gradient of host diffusate chemotactic factors. Positive chemotactic stimuli cause the J2 to orientate and migrate towards the host plant. We present validation data collected from assays of the root-knot nematode, Meloidogyne incognita, and the potato cyst nematode, Globodera pallida, which indicate a highly significant positive attraction of J2 of both species to respective host plants. This represents a simple, quick and inexpensive method of assessing host-finding behaviour in the laboratory. We consider that the pipette-bulb assay improves on previous host-finding/chemo-attraction assays through creating a more biologically relevant environment for experimental J2; analysis is quick and easy, allowing the straightforward interpretation of results. In addition, we have developed an 'agar trough' sensory assay variant which we believe can be used rapidly to ratify nematode responses to chemical gustatory or olfactory cues. This was constructed from a water agar substrate such that two counting wells were connected by a raised central trough, all flooded with water. Two small water agar plugs were dehydrated briefly in an oven and then hydrated in either an attractant, repellent or water control; these plugs were then placed in the terminal counting wells and subsequently leached the attractant or repellent to form a concentration gradient along the central trough, which contained the initial J2 innoculum. Our data show that both M. incognita and G. pallida J2 are positively attracted to host diffusates. In addition, they displayed a strong repulsion in response to 1 M NaCl2. J2 of M. incognita displayed a mild aversion to a non-host oak root diffusate, whereas G. pallida J2 displayed a strong aversion to the same non-host diffusate; neither species responded to a compost leachate. We believe that the agar trough assay improves on previous methods by facilitating rapid diffusion of attractant or repellents. Both of the aforementioned assays were designed as tools to assess the impact of RNAi-based reverse genetics screens for gene targets involved in chemosensory orientation.

FMRFamide-related peptides (FaRPs) in nematodes: Occurrence and neuromuscular physiology

The occurrence of classical neurotransmitter molecules and numerous peptidic messenger molecules in nematode nervous systems indicate that although structurally simple, nematode nervous systems are chemically complex. Thus far, studies on one nematode neuropeptide family, namely the FMRFamide-related peptides (FaRPs), have revealed an unexpected variety of neuropeptide structures in both free-living and parasitic species. To date 23 nematode FaRPs have been structurally characterized including 12 from Ascaris suum, 8 from Caenorhabditis elegans, 5 from Panagrellus redivivus and 1 from Haemonchus contortus. Ten FaRP-encoding genes have been identified in Caenorhabditis elegans. However, the full complement of nematode neuronal messengers has yet to be described and unidentified nematode FaRPs await detection. Preliminary characterization of the actions of nematode neuropeptides on the somatic musculature and neurones of A. suum has revealed that these peptidic messengers have potent and complex effects. Identified complexities include the biphasic effects of KNEFIRFamide/KHEYLRFamide (AF1/2; relaxation of tone followed by oscillatory contractile activity) and KPNFIRFamide (PF4; rapid relaxation of tone followed by an increase in tone), the diverse actions of KSAYMRFamide (AF8 or PF3; relaxes dorsal muscles and contracts ventral muscles) and the apparent coupling of the relaxatory effects of SDPNFLRFamide/SADPNFLRFamide (PF1/PF2) to nitric oxide release. Indeed, all of the nematode FaRPs which have been tested on somatic muscle strips of A. suum have actions which are clearly physiologically distinguishable. Although we are a very long way from understanding how the actions of these peptides are co-ordinated, not only with those of each other but also with those of the classical transmitter molecules, to control nematode behaviour, their abundance coupled with their diversity of structure and function indicates a hitherto unidentified sophistication to nematode neuromuscular intergration.

flp-32 Ligand/Receptor Silencing Phenocopy Faster Plant Pathogenic Nematodes

Restrictions on nematicide usage underscore the need for novel control strategies for plant pathogenic nematodes such as Globodera pallida (potato cyst nematode) that impose a significant economic burden on plant cultivation activities. The nematode neuropeptide signalling system is an attractive resource for novel control targets as it plays a critical role in sensory and motor functions. The FMRFamide-like peptides (FLPs) form the largest and most diverse family of neuropeptides in invertebrates, and are structurally conserved across nematode species, highlighting the utility of the FLPergic system as a broad-spectrum control target. flp-32 is expressed widely across nematode species. This study investigates the role of flp-32 in G. pallida and shows that: (i) Gp-flp-32 encodes the peptide AMRNALVRFamide; (ii) Gp-flp-32 is expressed in the brain and ventral nerve cord of G. pallida; (iii) migration rate increases in Gp-flp-32-silenced worms; (iv) the ability of G. pallida to infect potato plant root systems is enhanced in Gp-flp-32-silenced worms; (v) a novel putative Gp-flp-32 receptor (Gp-flp-32R) is expressed in G. pallida; and, (vi) Gp-flp-32R silenced worms also display an increase in migration rate. This work demonstrates that Gp30 flp-32 plays an intrinsic role in the modulation of locomotory behaviour in G. pallida, and putatively interacts with at least one novel G-protein coupled receptor (Gp-flp-32R). This is the first functional characterisation of a parasitic nematode FLP-GPCR. © 2013 Atkinson et al.

Anthelmintic resistance in Northern Ireland (II): Variations in nematode control practices between lowland and upland sheep flocks

A questionnaire to obtain information on nematode control practices and sheep management was sent to over 1000 farmers in Northern Ireland. Replies were received from 305 flock owners, and data from 252 of them were analysed. Farms were divided into lowland and upland areas. Sizes of pasture and stocking rates on lowland and upland farms were 59.5 hectares, 6.99 sheep/hectare and 62.9 hectares and 10.01 sheep/hectare, respectively. Mean drenching rates for lambs and adults were 2.33 and 2.44, respectively, in lowland flocks and 2.73 and 2.71, respectively, in upland flocks. Between 2008 and 2011, the most frequently identified compounds in use were benzimidazoles and moxidectin in lowland flocks, and benzimidazoles and avermectins in upland flocks. Over the same period the most frequently identified commercial formulations were Tramazole (R), Panacur (R) and Allverm (R) (white drench), Levacide (R) (yellow drench), Oramec (R) (clear drench; avermectin), Cydectin (R) (clear drench; moxidectin) and Monepantel (R) (orange drench).

Most respondents (56.35%) treated their lambs at weaning and the most common time to treat ewes was identified to be pre-mating (67.86% of respondents).

The results of the questionnaire survey revealed that lowland annual drench frequency was 233 and 2.44 in lambs and ewes, respectively, although drench frequencies were higher in upland flocks: 2.73 and 2.71 for lambs and ewes, respectively.

Annual drench rotation was practiced by 43.96% of flock owners, but whether this was true rotation or pseudo-rotation (i.e., substitution of one anthelmintic product by another product belonging to the same chemical group of anthelmintics) could not be explicitly determined. (C) 2012 Elsevier B.V. All rights reserved.

New insights into the FLPergic complements of parasitic nematodes: Informing deorphanisation approaches

FMRFamide-like peptide (FLP) receptors are appealing as putative anthelmintic targets. To date, 31 flp-encoding genes have been identified in Caenorhabditis elegans and thirteen FLP-activated G-protein coupled receptors (FLP-GPCRs) have been reported. The lack of knowledge on FLPs and FLP-GPCRs in parasites impedes their functional characterisation and chemotherapeutic exploitation. Using homology-based BLAST searches and phylogenetic analyses this study describes the identification of putative flp and flp-GPCR gene homologues in 17 nematode parasites providing the first pan-phylum genome-based overview of the FLPergic complement. These data will facilitate FLP-receptor deorphanisation efforts in the quest for novel control targets for nematode parasites.

Factors affecting biological control effectiveness of Pasteuria penetrans in Meloidogyne javanica and the bacterial development in the nematode body

The invasion and infectivity of Meloidogyne javanica juveniles (J2) encumbered with spore of Pasteuria Penetrans were influenced by the temperature and the time J2 were in the soil before exposure to roots. The percentage of infected females decreased as the time juveniles spent in soil increased. When spore encumbered J2 were maintained at 30 degrees C the decrease in infection was greater than that at 18 degrees C. The thermal time requirements and the base temperature for P. penetrans development were estimated. The rate of development followed an exponential curve between 21 and 36 degrees C and the base temperature for development was estimated by extrapolation to be 18.5 degrees C. The effect of integrating a nematode resistant tomato cultivar with the biocontrol agent P. penetrans also was investigated. The ability of the biocontrol agent to reduce numbers of root-knot nematodes was dependent on the densities of the nematode and P. penetrans spores in the soil.

The potential of combining Pasteuria penetrans and neem (Azadirachta indica) formulations as a management system for root-knot nematodes on tomato

Initial applications of 10(4) spores g(-1) of Pasteuria penetrans, and dried neem cake and leaves at 3 and 2% w:w, respectively, were applied to soil in pots. Juveniles of Meloidogyne javanica were added immediately to the pots (500, 5,000 or 10,000) before planting 6-week-old tomato seedlings. The tomatoes were sampled after 64 days; subsequently a second crop was grown for 59 days and a third crop for 67 days without further applications of P. penetrans and neem. There was significantly less root-galling in the P. penetrans combined with neem cake treatment at the end of the third crop and this treatment also had the greatest effect on the growth of the tomato plants. At the end of the third crop, 30% of the females were infected with P. penetrans in those treatments where spores had been applied at the start of the experiment. The effects of neem leaves and neem cake on the nematode population did not persist through the crop sequences but the potential for combining the amendments with a biological control agent such as P. penetrans is worthy of further evaluation.

Efficacy of neem (Azadirachta indica) formulations on biology of root-knot nematodes (Meloidogyne javanica) on tomato

Second stage juveniles of Meloidogyne javanica were exposed to aqueous extracts of neem crude formulations (leaves and cake) at 10%, 5%, and 2.5% w/v and a refined product, Aza at 0.1% w/v. The 10% extracts of neem leaf and cake caused 83% and 85% immobility and 35% and 28% mortality, respectively. Aza caused neither immobility or mortality of juveniles. When egg masses were placed in extracts of these formulations, hatching did not occur at all the concentrations (10%, 5%, 2.5% and 1.25% w/v) of the crude formulations. When the treated egg masses were returned to water, the eggs resumed hatching. Aza did not affect the nematode hatching. In glasshouse experiments, soil application of neem formulations significantly reduced the invasion of tomato roots by root-knot nematodes but once the nematodes managed to invade them, no effect detected on their development. Soil applications of Aza at 0.05% and 0.1% w/v significantly reduced the invasion and delayed development of nematodes within tomato roots whereas 0.025% did not. There were significantly fewer egg masses on tomato roots exposed to single egg mass in neem amended soil as compared to control. (C) 2007 Elsevier Ltd. All rights reserved.

Systemic and persistent effect of neem (Azadirachta indica) formulations against root-knot nematodes, Meloidogyne javanica and their storage life

Neem leaves, neem cake (a by-product left after the extraction of oil from neem seed) and a commercially refined product aza (azadirachtin) extracted from seed were evaluated. Aqueous extracts of crude neem formulations used as a seedling dip treatment significantly reduced the number of females and egg masses in roots whereas the refined one did not. A split-root technique was used to demonstrate the translocation of active compounds within a plant and their subsequent effect on the development of nematodes. When applied to the root portion all formulations significantly reduced the number of egg masses and eggs per egg mass. Whereas on the untreated root portion, neem cake at 3% w/w and aza at 0.1% w/w significantly reduced the number of egg masses as compared with neem leaves at 3% w/w, aza at 0.05% and control. All the neern formulations significantly reduced the number of eggs per egg mass on' the untreated root portion. The effect of neem leaves and cake on the development of root-knot nematodes was tested at 2, 4, 6, 8, and 16 weeks after their application to soil. Even after 16 weeks all the treatments significantly reduced the galling index and number of egg masses but their effectiveness declined over time. After storing neem leaves, cake and aza for 8 months under ambient conditions the efficacy of neem leaves and aza, against root-knot nematodes, remained stable whereas that of cake declined. (c) 2006 Elsevier Ltd. All rights reserved.

Genotypic differences in the growth of bananas (Musa spp.) infected with migratory endoparasitic nematodes. 2. Shoots

In order to identify the effect of burrowing nematodes on the shoots (pseudostem and leaves) of banana plants and to determine whether or not shoot characteristics are associated with plant resistance to nematodes two experiments were conducted in controlled conditions within polytunnels. The banana plants were harvested on three occasions for the measurement of root morphology and biomass. Varieties differed in their resistance to nematodes from resistant (Yg Km5, FHIA 17, FHIA 03) and partly resistant (FHIA 01, FHIA 25) to not resistant (FHIA 23, Williams). Nematodes reduced total plant dry weight at the first harvest in Experiment 1 and by an average of 8.8% in Experiment 2, but did not affect leaf area in either experiment. The ratio of above-ground Weight to total plant weight was reduced from 75% to 72% in nematode-infected plants compared with the control plants for all varieties tested in Experiment 1, but was only reduced in FHIA 25 and FHIA 23 in Experiment 2. Varieties differed in above-ground growth. The FHIA varieties had greater shoot weights and leaf area than YgKm5 and Williams. Overall, resistance to nematodes was associated with the partitioning of a greater proportion of biomass to the roots than to above-ground parts.

A comparative study on the effectiveness of laboratory bioassays of entomopathogenic nematodes against desert locust nymphs Schistocerca gregaria (Acrididae:Orthoptera)

Entomopathogenic nematodes, Steinernema carpocapsae, S. feltiae (Steinernematids) Heterorhabditis indica and H. bacteriophora (Heterorhabditids) were studied to control nymphs of desert locust Schistocerca gregaria. Results of all experiments showed a significant difference in mortality percentage among all isolates. All nematodes were found more effective when exposure time was increased up to 10 days. On the other hand, both Heterorhabditids caused maximum mortality as compared to Steinernematids at 30 degree C. When different moisture levels were tested in the sand arena, a medium level of moisture (1%) caused maximum insect mortality in all isolates. However, highest concentration of each isolate (200 IJs per ml) proved to be most appropriate for maximum insect death. Similarly, both Heterorhabditis nematodes when orally applied to insects killed maximum nymphs as compared to other two Steinernematids. A similar response was observed in infectivity test when maximum percentage of IJs of both isolates of Heterorhabditis successfully penetrated into the body of locust nymphs. This research suggests some useful basic findings in developing biocides with suitable virulent of entomopathogenic nematode for controlling nymphs of desert locust.

Condensed tannins act against cattle nematodes

The use of natural plant anthelmintics was suggested as a possible alternative control of gastrointestinal nematodes (GIN) in ruminants. Direct anthelmintic effects of tannin-containing plants have already been shown in sheep and goat GIN. These anthelmintic properties are mainly associated with condensed tannins. In the present study, we evaluated possible in vitro effects of three tannin-containing plants against bovine GIN. Effects of Onobrychis viciifolia, Lotus pedunculatus and Lotus corniculatus condensed tannin (CT) extracts on Cooperia oncophora and Ostertagia ostertagi were determined by a larval feeding inhibition assay (LFIA) and a larval exsheathment assay (LEA). In the LFIA, all three plant extracts significantly inhibited larval feeding behaviour of both C. oncophora and O. ostertagi first stage larvae in a dose-dependent manner. The L. pedunculatus extract, based on EC50 (effective concentration for 50% inhibition), was the most effective against both nematodes, followed by O. viciifolia and L. corniculatus. The effect of CT extracts upon larval feeding behaviour correlates with CT content and procyanidin/prodelphidin ratio. Larval exsheathment of C. oncophora and O. ostertagi L3 larvae (third stage larvae) was also affected by CT extracts from all three plants. In both in vitro assays, extracts with added polyvinylpolypyrrolidone, an inhibitor of tannins, generated almost the same values as the negative control; this confirms the role of CT in the anthelmintic effect of these plant extracts. Our results, therefore, indicated that tannin-containing plants could act against cattle nematodes.

Techniques for image analysis of movement of juveniles of root-knot nematodes encumbered with Pasteuria penetrans spores

Root-knot nematodes (Meloidogyne spp.) are the most significant plant-parasitic nematodes that damage many crops all over the world. The free-living second stage juvenile (J2) is the infective stage that enters plants. The J2s move in the soil water films to reach the root zone. The bacterium Pasteuria penetrans is an obligate parasite of root-knot nematodes, is cosmopolitan, frequently encountered in many climates and environmental conditions and is considered promising for the control of Meloidogyne spp. The infection potential of P. penetrans to nematodes is well studied but not the attachment effects on the movement of root-knot nematode juveniles, image analysis techniques were used to characterize movement of individual juveniles with or without P. penetrans spores attached to their cuticles. Methods include the study of nematode locomotion based on (a) the centroid body point, (b) shape analysis and (c) image stack analysis. All methods proved that individual J2s without P. penetrans spores attached have a sinusoidal forward movement compared with those encumbered with spores. From these separate analytical studies of encumbered and unencumbered nematodes, it was possible to demonstrate how the presence of P. penetrans spores on a nematode body disrupted the normal movement of the nematode.

In vitro effects of extracts and purified tannins of sainfoin (Onobrychis viciifolia) against two cattle nematodes

Sainfoin (Onobrychis viciifolia) is a condensed tannin (CT)-containing legume and has anthelmintic potential against gastrointestinal nematodes of ruminants. This study investigated in vitro effects of acetone/water extracts and derived CT fractions from different types of sainfoin (i.e. accessions) against larvae of Cooperia oncophora and Ostertagia ostertagi by applying the larval feeding inhibition assay (LFIA). Seven sainfoin accessions were extracted and tested with L1 larvae at 10 and 40 μg extract/ml. In addition, CT in extracts from 4 accessions were fractionated according to polymer size and tested by LFIA at two concentrations (2 and 10 μg CT fraction/ml). All sainfoin extracts caused significant inhibition of L1-feeding of both C. oncophora and O. ostertagi with varying intensity compared to the control (phosphate buffered saline). For both nematode species the in vitro effect was positively correlated with CT content in the extracts, but not with any of the structural CT parameters. In contrast, the 16 CT fractions revealed significant correlations between in vitro effect and CT content, polymer size (mean degree of polymerisation, mDP) and monomeric composition (prodelphinidin percentage, % PD). These differences between crude extracts and purified fractions may stem from the fact that extracts contain complex CT mixtures, which may mask and thus suppress CT structural effects. This study provides the first indication that, apart from CT and % PD content, polymer size also contributes to anthelmintic activity of CTs. The results, therefore, suggest that the inter-accession variability in CT content and composition needs to be taken into account in future plant breeding programmes which seek to enhance the anthelmintic properties of sainfoin