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Published as doi: 10.1096/fj.06-7343com.
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(The FASEB Journal. 2007;21:1233-1243.)
© 2007 FASEB

flp gene disruption in a parasitic nematode reveals motor dysfunction and unusual neuronal sensitivity to RNA interference

Michael J. Kimber*, Susan McKinney{dagger}, Steven McMaster{dagger}, Tim A. Day*, Colin C. Fleming{ddagger} and Aaron G. Maule{dagger},1

* Department of Biomedical Sciences, Iowa State University, Ames, Iowa, USA;

{dagger} Parasitology, School of Biological Sciences, Queen’s University Belfast, Belfast, UK; and

{ddagger} Agri-Food and Biosciences Institute, Belfast, UK

1Correspondence: School of Biological Sciences, Queen’s University Belfast, Medical Biology Centre, 97 Lisburn Rd., Belfast BT9 7BL, UK. E-mail: a.maule{at}qub.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The potato cyst nematode Globodera pallida is a serious pest of potato crops. Nematode FMRFamide-like peptides (FLPs) are one of the most diverse neuropeptide families known, and modulate sensory and motor functions. As neuromuscular function is a well-established target for parasite control, parasitic nematode FLP signaling has significant potential in novel control strategies. In the absence of transgenic parasitic nematodes and the reported ineffectiveness of neuronal gene RNAi in Caenorhabditis elegans, nothing is known about flp function in nematode parasites. In attempts to evaluate flp function in G. pallida, we have discovered that, unlike in C. elegans, these genes are readily susceptible to RNAi. Silencing any of the five characterized G. pallida flp genes (Gp-flp-1, -6, -12, -14, or -18) incurred distinct aberrant behavioral phenotypes consistent with key roles in motor function. Further delineation of these effects revealed that double-stranded RNA exposure time (≥18 h) and concentration (≥0.1 µg/ml) were critical to the observed effects, which were reversible. G. pallida flp genes are essential to coordinated locomotory activities, do not display redundancy, and are susceptible to RNAi, paving the way for the investigation of RNAi-mediated flp gene silencing as a novel plant parasite control strategy.—Kimber, M. J., McKinney, S., McMaster, S., Day, T. A., Fleming, C. C., Maule, A. G. flp gene disruption in a parasitic nematode reveals motor dysfunction and unusual neuronal sensitivity to RNA interference.


Key Words: FMRFamide-like peptide • neuropeptide • nervous system • locomotion • helminth parasite


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PLANT PARASITIC NEMATODES (PPNS) cause vast economic losses wherever crop plants are grown, with estimates suggesting annual yield losses in excess of $100 billion in U.S. dollars (1 , 2) . Concerns over environmental safety have caused the deregistration of several frontline nematicides, leading to serious shortfalls in the efficacy of current pest management strategies and a pressing need for novel methods of plant-parasitic nematode control. Efforts to plug these gaps include the development of transgenic plants that can resist the establishment and/or maintenance of parasitic nematode infections (3) . To this end, several groups have shown that PPNs are susceptible to RNA interference (RNAi) such that transgenic strategies based on the in planta generation of nematode gene-specific, double-stranded RNA (dsRNA) hold some promise (4 5 6 7 8 9) . Recently, tobacco plants transformed using Agrobacterium to express dsRNA for splicing factor or integrase from the root knot nematode Meloidogyne incognita showed significant resistance to parasite infection (10) , thus confirming the utility of these approaches.

Of critical relevance to PPN survival is a viable neuromuscular system that permits plant location, penetration, secretory activities, migration, alimentation, and reproduction. Within animal parasitic nematodes, most of the leading antiparasite drugs (anthelmintics) act on targets within the neuromuscular system; recently, neuropeptide signaling systems involved in regulating motor function have triggered significant interest as novel drug targets in metazoan parasites (11 12 13) . The largest family of neuropeptides in nematodes are the FMRFamide-like peptides (FLPs), which possess a C-terminal Arg-Phe-NH2 signature and play a central role in motor activities. Studies of the model species Caenorhabditis elegans suggest that the number of FLPs present in nematodes is staggering, with ~28 flp genes encoding at least 72 distinct peptides (14 15 16) ; all of these flp genes (and several others not identified in C. elegans) are represented within the current complement of parasitic nematode-expressed sequence tags (ESTs), 21 of them within the PPN ESTs (16) .

Several PPN flp genes have been fully characterized, and FLPs have been shown to be widely expressed in the nervous systems of the cyst-forming nematodes Heterodera glycines, Globodera pallida, and Globodera rostochiensis (17 , 18) . In C. elegans, some flp gene knockouts have induced multiple aberrant behavioral phenotypes (19) , and each flp gene is expressed in a unique set of neurons (20) consistent with FLP roles in a wide array of different motor behaviors. Although flp expression patterns in PPNs do not appear to mirror those seen in C. elegans, a similar restricted and unique neuronal expression pattern was reported for each flp gene examined (21) . Currently there are no functional data for PPN FLPs.

The well-established utility of nematode motor function as a target for anthelmintic drugs and the discovery that PPNs are susceptible to RNAi raise the possibility that individual FLPs and/or components of FLP signaling, if susceptible to gene silencing methods, could provide an attractive target for transgenic approaches to plant parasite control. However, a number of distinct observations highlight potential barriers to the exploitation of flp gene RNAi for PPN control. First, the infective (J2) larvae of PPNs are nonfeeding, and the in vitro induction of RNAi has been successful only where feeding activity has been triggered using the classical transmitter, octopamine, or the crystalline phenol, resorcinol (4 , 9) . Since entry into the plant will stimulate feeding activity in infecting larvae, this problem is unlikely to impede the in vivo uptake of plant-derived dsRNA. Second, although individual FLPs have broad-ranging and profound activities in nematode bioassays, many flp gene knockouts show no observable phenotype in C. elegans (14) . Finally, many studies report the refractory nature of neuronally expressed genes in C. elegans to RNAi (22 23 24 25 26) . As a result, hairpin transgenes and/or additional mutations in the rrf-3 (which encodes a putative RNA-directed RNA polymerase that negatively regulates RNAi) or eri-1 (which encodes an RNase that can degrade dsRNA) genes are needed to enhance the susceptibility of many neuronal genes to RNAi-based silencing procedures (27 28 29 30) .

The potato cyst nematode, G. pallida, and its sibling species, G. rostochiensis, are the most significant helminth pests of the potato industry. In the present study we report the sensitivity of neuronally expressed flp genes in nonfeeding G. pallida J2s to RNAi, findings that are in direct contrast to observations in C. elegans and other PPNs. Furthermore, we show that silencing of five different flp genes triggers motor dysfunction in a manner that highlights flp gene potential as a target for the control of plant parasitic nematodes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
G. pallida (pathotype Pa2/3) were cultured on potato cv Cara at the Nematology Laboratory, Department of Agriculture and Rural Development for Northern Ireland (Belfast). Infective stage juveniles (J2) were hatched from cysts using potato root diffusate at room temperature (31) , then washed thoroughly in diethyl pyrocarbonate (DEPC)-treated water.

dsRNA synthesis
Poly(A)+ RNA was extracted from ~30 µl (packed volume) freshly hatched J2 G. pallida using Dynabeads mRNA DIRECT Kit (Dynal, Great Neck, NY, USA) as described by the manufacturer. This RNA was used to synthesize a single-stranded cDNA polymerase chain reaction (PCR) template using the SMART RACE cDNA Amplification Kit (BD Biosciences, Bedford, MA, USA). Oligonucleotides were designed to amplify distinct regions of selected G. pallida flp genes (see Fig. 1 ), so that for each region two templates were generated from cDNA: one amplicon with the T7 polymerase promoter sequence at the 5' end of the coding strand and the other with the promoter site at the 5' end of the noncoding strand. These RNA transcription templates were gel purified and used to synthesize the corresponding coding or noncoding RNA strand using the T7 MEGAscript Kit (Ambion, Austin, TX, USA). Purified transcription products were analyzed spectrophotometrically at 260 nm to quantify RNA. Double-stranded RNA was assembled by incubating equimolar amounts of each strand at 68°C for 10 min, followed by 30 min at 37°C; subsequent treatment with DNase removed template. Gel electrophoresis of the products confirmed formation of a double-stranded construct, that the constructs were of the correct size, and also confirmed previous quantification.


Figure 1
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  		Figure 1. Illustration of the Gp-flp open reading frames (ORFs) and the regions with which the double-stranded RNAs (gray) used in this study have homology. Solid black boxes represent the relative positions of the encoded peptides.

Nematode treatment and knockout analysis using RT-polymerase chain reaction (RT-PCR)
Around 250 freshly hatched J2 worms were incubated for the desired length of time in 100 µl of each dsRNA construct diluted with DEPC-treated water to the required final concentration. For controls, worms were also incubated in DEPC-treated water or concentration- and size-matched dsRNA derived from a non-nematode gene (see below). Incubations were performed at room temperature in hydrophobically lined, 1.5 ml microcentrifuge tubes (Anachem, Luton, Beds, UK). Postincubation, worms were washed thoroughly three times in DEPC-treated water before total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The RNA was isopropanol precipitated, washed in 75% ethanol, and resuspended in 20 µl DEPC-treated water. After DNase treatment, the RNA was used in first-strand cDNA synthesis with the First-Strand Synthesis System (Invitrogen) driven by an oligo dT primer. This cDNA served as a template in a PCR reaction to amplify the FLP-encoding gene under investigation with primers flanking the region of the gene homologous to the dsRNA.

Analysis of knockdown phenotype
Nematodes were incubated in dsRNA as described for the desired length of time, with worms incubated in sterile, DEPC-treated water serving as a negative control. Analysis of aberrant nematode locomotion using a migration assay constituted the primary means of detecting knockdown phenotype. Approximately 100 treated and control juveniles were added to the top of moistened sand columns made by filling a 5 cm length of glass tubing (5 mm internal diameter) with washed, coarse sand and covering the base of the tube with nylon muslin. The columns were placed vertically in collection vials containing sufficient water to cover the base of the column. The numbers of worms migrating through the columns and into the collection vials were counted over a 12 h period at 2 h increments. After the 12 h time course, columns were washed through with water and any nematodes incapable of completing the migration were recovered and counted. One-way ANOVA, Wilcoxon Sum Test analysis, and least significant difference tests were used to analyze the results of the assay.

Further, the posture and activity of those nematodes incubated in dsRNA were compared with worms incubated in water in an attempt to discern any atypical behavior or irregular movement.

Changes to flp gene nomenclature
We also propose a change in nomenclature for those genes examined here and which we discovered and characterized previously (18) . Originally, the five G. pallida flp genes were designated chronologically upon identification, hence Gp-flp-1 through Gp-flp-5. However, the degree of conservation in phylum Nematoda is remarkable, such that all of the G. pallida flp genes have homologues in C. elegans and many other nematode species. We therefore propose to adopt a nomenclature that is based on the original C. elegans gene designations, so that Gp-flp-1 through Gp-flp-5 should now be referred to as Gp-flp-6, Gp-flp-12, Gp-flp-14, Gp-flp-1, and Gp-flp-18, respectively.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gp-flp genes can be silenced using RNAi
Our initial experiments investigating the susceptibility of G. pallida flp genes to RNAi focused on Gp-flp-6, which encodes four copies of the FLP, KSAYMRFamide. This gene was chosen because earlier in situ hybridization studies revealed a more extensive distribution within the G. pallida nervous system than for the other flp genes (21) . Worms were incubated for 24 h in one of three Gp-flp-6 dsRNA constructs of varying lengths (88 bp, 227 bp, and 316 bp) corresponding to three different regions of the Gp-flp-6 open reading frame (Fig. 1) . The dsRNA was diluted with DEPC-treated water to a final concentration of 0.1 mg/ml. To confirm Gp-flp-6 silencing, an RT-PCR was performed on poly(A)+ RNA extracted from worms incubated in each of the three dsRNA constructs. The primers used in this PCR reaction were designed to anneal upstream and downstream of the RNA assemblies and to amplify a 588 bp region of Gp-flp-6. This amplicon was generated from cDNA obtained from the positive control worms (incubated in DEPC-treated water) and from those incubated in the 88 bp dsRNA construct, but could not be amplified from the nematodes incubated in either the 227 bp or 316 bp dsRNA constructs (Fig. 2 A). On a few occasions a very faint amplicon could be seen after incubation in the 316 bp dsRNA (not visible in Fig. 2A ). Both to confirm the quality of all cDNA obtained and to test the specificity of Gp-flp-6 knockdown, we attempted to amplify the Gp-flp-1 gene (18) from all our cDNAs, including our RNAi experimental worms. We were able to successfully amplify this gene from all of our cDNA preparations (results not shown). Thus, the Gp-flp-6 knockdown was deemed gene specific.


Figure 2
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  		Figure 2. A) RT-PCR reactions amplifying 588 bp Gp-flp-6 fragment using templates of cDNA constructed from RNA extracted from Globodera pallida J2 incubated in 0.1 mg/ml dsRNA constructs. Lane 1: nematodes incubated in water; lanes 2–4: nematodes incubated in double-stranded (ds)RNA of lengths 316 bp, 227 bp, and 88 bp, respectively; lane 5: negative RT-PCR control (no template). No fragment was amplified from nematodes incubated in 316 bp and 227 bp dsRNA, indicating effective gene silencing. B) Motility of G. pallida J2 after 24 h incubation in varying length fragments of dsRNA homologous to Gp-flp-6. Worm motility was evaluated using a sand column functional migration assay. Incubation period in dsRNA was increased to 2 days (C) and 7 days (D). Control worms were incubated in DEPC-treated water. Results are presented as mean +SE (n≥3).

Silencing of Gp-flp-6 disrupts normal locomotory behavior
Many of those FLPs tested in physiological assays are potently myoactive. Our hypothesis was that the Gp-flp-6 silenced nematodes would display abnormal locomotory behavior and, as a consequence, reduced motility. Worms incubated in the two effective dsRNA constructs (227 bp and 316 bp) for 24 h, 2 days, and 7 days were assessed for impaired motility using a migration assay whereby nematodes migrate the length of a 5 cm sand column. Under normal conditions, nematodes migrate downward through the sand column; worms with disrupted motor function have an impaired ability to complete this migration. When nematodes were incubated for 24 h in DEPC-treated water, a maximum mean migration of 75.8% (n=4) was recorded in a 12 h period (see Fig. 2B ). By comparison, worms incubated in the dsRNA constructs showed significant inhibition of motility (n=4, P=0.01). After 12 h, 28.7% of the juveniles incubated in the 316 bp Gp-flp-6 dsRNA and 12.2% of those incubated in the 227 bp Gp-flp-6 dsRNA had migrated through the column representing a 62.1% and 83.9% inhibition of normal worm motility, respectively, compared with control worms. At each 2 h time point throughout the assay, the inhibition of motility observed upon incubation in the 227 bp dsRNA appeared greater than that of the 316 bp dsRNA. However, this disparity was not significant. The inhibition detected after 24 h incubation was also apparent after 2 days of incubation in each dsRNA construct, but this time the inhibition of motility was found to be more profound (n=4, P=0.01). After 12 h, nematode migration through the column was 16.1% and 2.0% for the 316 bp and 227 bp dsRNA, respectively, constituting a 76.4% and 97.0% reduction in normal worm migration behavior. Again, the shorter dsRNA fragment appeared more potent, but this difference was not significant. After 7 days of incubation in the dsRNA constructs, the effect of gene silencing was most profound, with 7.3% (316 bp) and 3.0% (227 bp) of worms completing the migration (n=4, P=0.01), translating to a 88.4% (316 bp) and a 95.2% (227 bp) inhibition of normal worm activity. As with the 1 and 2 day incubations, the shorter dsRNA seemed to be more effective, but differences were not significant. Based on these observations, dsRNA constructs of ~220–230 bp were selected for all subsequent experiments on Gp-flp-6 and the other Gp-flp genes examined in this study (see Fig. 1 ).

Besides the migration assay, worms with silenced Gp-flp-6 were observed for any visual knockdown phenotype as revealed by atypical motile behaviors or posture. Under normal conditions, G. pallida J2 are active, motile worms displaying the typical nematode sinusoidal waveform. Worms incubated in the two dsRNA constructs for 24 h and 2 days appeared normal. However, worms incubated for 7 days appeared straight and rigid, a phenotype designated "straight." The appearance of this paralyzed phenotype after 7 days reflects the increasingly inhibited movement in the migration assay. RT-PCR demonstrated that overnight incubation in the dsRNA was sufficient to grossly affect the presence of Gp-flp-6 mRNA. Thus, the increased severity of knockdown phenotype over the course of a week may be the result of a peptide stored in synaptic vesicles at the nerve terminal being used up and not replenished. Also, although no visual knockdown phenotype could be detected after 24 h and 2 days, a profound inhibition of migratory behavior was observed over this same time frame. Clearly, normal motor function is being rapidly disrupted even without a discernable visual phenotype. This observation is also apparent with the other flp genes.

Silencing Gp-flp genes induces potent aberrant phenotypes
Having demonstrated that Gp-flp-6 could be silenced using RNAi by soaking and that silencing this gene grossly altered normal worm behavior, we investigated the effects of silencing additional G. pallida flp genes. The effects on worm migration of silencing genes Gp-flp-1, -12, -14, and -18, confirmed by RT-PCR, can be found in Table 1 (the effect of the 227 bp dsRNA for Gp-flp-6 is included for comparison). Silencing of any of the genes examined resulted in profound inhibition of migration through the sand column. The pattern established in preliminary Gp-flp-6 experiments—that of an increasing efficacy of inhibition correlating to the duration of dsRNA incubation—was not observed with Gp-flp-1, Gp-flp-12, and Gp-flp-18. Typically, the inhibition of motility had a more abrupt onset, and silencing of Gp-flp-1, Gp-flp-12, and Gp-flp-18 produced an almost complete inhibition of migratory behavior after the 24 h incubation. This knockdown effect compared with normal worm activity (control worms incubated in DEPC-treated water) ranged from 100% on Gp-flp-12 silencing (i.e., no worms completing migration) to 99.5% and 97.6% with Gp-flp-18 and Gp-flp-1 silencing, respectively. Silencing Gp-flp-14 produced an inhibitory effect more qualitatively similar to that of Gp-flp-6. 18.2% of worms completed the migration after the 24 h dsRNA incubation, a 70.7% decrease compared with control worms. Prolonged incubations of 2 and 7 days in dsRNA abolished worm migration.


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  		Table 1. Summary of the effects of Gp-flp gene RNAi on the locomotory activity of Globodera pallida infective (J2) larvaea

Observations of the worms exposed to dsRNA for these additional four Gp-flp genes yielded a number of distinct aberrant phenotypes. The phenotypes and their descriptors are summarized in Table 2 . Incubation in Gp-flp-12 dsRNA completely inhibited worm migration and generated a straight, paralyzed posture as the dominant phenotype after 24 h, 2 days, and 7 days. However, after 24 and 2 day incubations, a small number of worms appeared phenotypically normal, maintaining a sinusoidal body form; others appeared to display a slow phenotype, where the nematodes appeared normal but moved with markedly reduced speed (not quantified). Two further phenotypes were observed in Gp-flp-14 silenced nematodes. After 24 h, worms appeared either normal or stationary, a phenotype characterized by maintenance of the sinusoidal body form but with a lack of either forward or backward motility. As with the other gene knockouts, this phenotype progressed and a twitching phenotype was observed after 2 days of incubation. Twitching worms resembled stationary but with jerky, uncoordinated movement. After 7 days, all worms appeared straight. Gp-flp-1 knockout worms displayed the greatest range of abnormal phenotypes. After 24 h incubation in dsRNA, twitching was the predominant phenotype, which developed rapidly over the next 24 h. Those that did move appeared to have the slow phenotype, but this was sequentially replaced by a stationary and coiled posture whereby the nematodes would coil up, particularly toward their anterior, and remain inactive. After 7 days, the worms appeared straight. Finally, Gp-flp-18 silenced worms appeared slow on the 24 h and 2 day incubations but, like the other genes, were straight after 7 days. Note that we were able to amplify Gp-flp-6 using RT-PCR from worms soaked in dsRNA for any of the other four transcripts, further indicating the specificity of the silencing events (results not shown).


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  		Table 2. The most common phenotypes observed following Gp-flp gene RNAi in Globodera pallida infective (J2) juvenilesa

Effects of non-nematode dsRNA on worm locomotion and phenotype
Silencing each of the flp genes examined induced profound effects on nematode behavior. We wondered whether this might reflect some general toxic or inhibitory action of the dsRNA constructs rather than the specific silencing of genes critical to nematode neuromuscular function. To address this question, we incubated worms in dsRNA, which is not homologous to a nematode gene that may be involved in neuromuscular function. For this purpose, we chose a region of a chloroplast-specific ribosomal protein gene from the tomato, Lycopersicon esculentum (accession no. AY568722). We chose this DNA sequence primarily because it was unlikely to have homology to any nematode genes; indeed, no homologues were found using similarity searches of the G. pallida EST database or the C. elegans genome sequence. We found no significant difference in migratory behavior of worms incubated in L. esculentum dsRNA (concentration- and size-matched to effective Gp-flp gene dsRNAs) compared with worms incubated in water (results not shown). Taken with the RT-PCR confirmation that the targeted Gp-flp genes are no longer being expressed, this confirms that the suppressive phenotypes we observed are due to silencing of the flp genes under investigation rather than being caused by any nonspecific action of our dsRNA constructs.

Persistence of the Gp-flp gene silencing effects
The inhibition of nematode motility demonstrated here provides evidence of the critical importance of FLPs in maintaining normal neuromuscular function in PPNs. The data not only validate FLP-mediated neurotransmission as a target for PPN control strategies, but also indicate the potential of this RNAi-based approach for the control of these economically important parasites. With this in mind, we decided to dissect the observed response further.

To determine the durability of the motility knockdown, worms were incubated in Gp-flp-12 dsRNA for 24 h as before prior to being thoroughly washed with DEPC-treated water to remove the RNA before incubation in DEPC-treated water for either another 24 h or 6 days. As controls, worms were either incubated in DEPC-treated water for 2 or 7 days or incubated in Gp-flp-12 dsRNA for 2 or 7 days. All nematodes were then assessed using the migration assay to investigate whether the inhibition of motility continued in a prolonged absence of the dsRNA trigger. Gp-flp-12 was chosen for this experiment, as silencing this gene was deemed to have the greatest impact on worm motility. When we compared nematodes that had a 24 h recovery period in DEPC-treated water post-dsRNA incubation to ones that had been incubated in dsRNA continually for 48 h, we found that those with the recovery period showed improved migration through the sand column (7.7% worms migrating compared with 2.0%, see Table 3 ). However, this effect was not significant. With an increased recovery period of 6 days in DEPC-treated water, 27.1% of nematodes completed the sand column migration compared with only 4.1% of those worms that had been maintained in the dsRNA for 7 days. This difference was found to be significant (n=3, P<0.05) and demonstrates that the knockdown effect was wearing off. When expressed relative to the DEPC-treated water control (=normal worm behavior), nematode migration had returned to 39.4% of its normal level, compared with being only 6.0% of normal activity in the absence of a recovery period.


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  		Table 3. Recovery of locomotory abilities in Globodera pallida infective (J2) larvae after removal of Gp-flp-12 double-stranded RNAa

Concentration-dependent effects of Gp-flp RNAi
In previous studies that used RNAi to silence genes of interest in parasitic nematodes, disparate concentrations of dsRNA were used to suppress gene translation, ranging from 0.5 to 5 mg/ml (4 , 5 , 6 , 32 33 34 35 36) . There has been no specific investigation of any RNA concentration dependency associated with RNAi in parasitic nematodes, and consequently there is no information as to the concentration of dsRNA required to trigger silencing. We addressed this question by incubating nematodes in 10-fold serial dilutions of dsRNA, with concentrations between 0.1 mg/ml and 1 pg/ml for 24 h, then assaying for functional defects using the migration assay. Gp-flp-12 was again chosen due to the robustness of its effect; worms incubated for 24 h in dsRNA at 0.1 mg/ml showed a profound inhibition of motility (see Fig. 3 ). At concentrations between 10 µg/ml and 0.1 µg/ml, dsRNA worm migration was still significantly impaired. Nevertheless, worm migration increased from ~10% at 10 µg/ml dsRNA to ~20% at 0.1 µg/ml dsRNA, indicating a slowly increasing number of worms migrating successfully. The levels of worm migration increased dramatically at concentrations of dsRNA <0.1 µg/ml, with no significant reduction in worm migration being recorded after 24 h incubations in dsRNA concentrations between 10 ng/ml and 1 pg/ml. Note that worms incubated in dsRNA (0.1 mg/ml to 0.1 µg/ml) for 7 days showed a pattern of inhibition that mirrored the 24 h incubations (data not shown).


Figure 3
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  		Figure 3. Effective gene silencing was found to be dependent on RNA concentration. Worms were incubated in a range of concentrations of Gp-flp-12 dsRNA for either 24 h or 7 days before being assayed for aberrant motility using the migration assay; control worms were incubated in DEPC-treated water. Results are presented as mean +SE (n≥3).

Soaking duration effects on Gp-flp RNAi
Previous studies of the RNAi of selected genes in PPNs have involved incubating worms or egg-masses in dsRNA for differing periods (4 h to 3 days). Although Chen et al. (6) reported the importance of soaking for 24 h as opposed to the 4 h reported by Urwin et al. (4) , no one has reported the effects of varying the soaking duration. In this study, the minimum time that G. pallida J2s were soaked in Gp-flp-12 dsRNA was 24 h. Further reducing the length of time that worms were soaked in 0.1 mg/ml Gp-flp-12 dsRNA was found to markedly reduce the efficacy of the dsRNA trigger in inducing RNAi (see Table 4 ). Although a small reduction in the percentage of worms migrating was seen after a 6 h soak (88.6% of control migration), the effects were not significant (n=3, P>0.05). Significant effects were seen after 12 h and 18 h soaks in dsRNA, with the number of treated worms completing the migration falling to 70.9 ± 1.9% (n=4, P<0.05) and 59.6 ± 2.3% (n=5, P<0.05) of control, respectively. The differences in the level of migration seen after 18 h and 24 h soaks were profound, with a ~55% drop in the level of worm migration with an extra 6 h soak time. These data indicate that soaking duration is critical to the observed phenotypic abnormalities induced by Gp-flp dsRNA.


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  		Table 4. The effects of Gp-flp-12 double-stranded RNA (0.1 mg/ml) soaking duration on the locomotory ability of Globodera pallida infective (J2) larvaea

Octopamine effects on the locomotion of Globodera pallida infective (J2) larvae
Other workers have shown that the induction of feeding by, for example, octopamine is required to facilitate the uptake of dsRNA by PPNs (4) . We wanted to examine whether octopamine could enhance the observed effects of Gp-flp-12 dsRNA on worm locomotory capabilities. However, we found that incubating G. pallida infective (J2) larvae in 50 mM octopamine (as previously used for this purpose) inhibited worm motility and therefore prevented analysis of RNAi-induced locomotory defects. The enhancement of pharyngeal pumping by octopamine reported earlier was observed.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study uses RNAi by soaking to examine the function of neuropeptide-encoding flp genes in the infective stages (J2) of the nematode parasite, G. pallida. Soaking C. elegans in dsRNA has proved to be an effective method of delivery and has advantages when applied to the study of plant parasitic nematodes, as their small size makes microinjection difficult; since they do not ingest until they have established a feeding site in host plant tissues, feeding approaches to dsRNA delivery (22 , 37 , 38) seemed to be impossible. This latter problem was overcome by Urwin and colleagues (4) , who found that incubation of J2 G. pallida and the related soybean cyst nematode, Heterodera glycines, in 50 mM octopamine stimulated pharyngeal pumping and promoted worm ingestion of exogenous dsRNA. The presence of octopamine was a prerequisite for effective RNAi gene silencing. In contrast, in the present study we have found that potent silencing of flp genes can be effected in the absence of octopamine. Indeed, when a protocol for silencing involving 50 mM octopamine was used, we found it was pointedly disadvantageous, since at this concentration octopamine rendered the worms incapable of normal migratory behaviors, masking the silencing effects of our target genes; this does not rule out the possibility that octopamine could further enhance flp silencing in PCN. Silencing of flp genes in the present study could be achieved simply by incubating the nematodes in water containing dsRNA. If octopamine is required for oral RNA uptake, this suggests that an alternative mechanism by which the RNA is gaining systemic access is operating here, possibly via the sensory structures (amphids), the secretory/excretory pore, or even the cuticle.

The expression patterns of four of the five flp genes whose function we have investigated in the present study had previously been delineated using in situ hybridization (21) . Each mRNA, the target for the gene silencing observed here, has a highly localized distribution within the nervous system of G. pallida J2, being expressed in the soma of cells innervating nematode musculature and sensory structures as well as those of interneurones. A most significant finding of the present study is that these neuronally expressed genes can be silenced readily by using a simple soaking protocol. Although the cellular machinery by which the RNAi phenomenon operates is clearly present in nematode neuronal tissue (23 , 39) , neuronal targets have commonly proved to be refractory to the process (22 , 25 , 26) . Indeed it has required the identification and subsequent use of rrf-3 or eri-1 mutant C. elegans, strains hypersensitive to RNAi, before many neuronal genes could be successfully silenced (25 , 28 , 40) or the lack of phenotypes detected. Why RNAi silences the neuronally expressed flp genes so readily in G. pallida, and not neuronal targets in C. elegans, is unclear, but could relate to differential cellular/neuronal distributions of RRF-3 (26) .

Flp gene silencing in G. pallida appears to depend on the concentration, exposure time, and (at least for Gp-flp-6) size of exogenous dsRNA. Suppressive effects were observed at RNA concentrations as low as 0.1 µg/ml; for Gp-flp-6, short RNA assemblies (88 bp) had little effect whereas longer fragments (227–316 bp) possessed far superior silencing capabilities. This size dependency has been reported in a parasitic protistan (41) . A linear relationship between RNA length and a log value of inhibition between 80 and 900 bp isapparent in Drosophila embryos (42) , indicating a disproportionate increase in effect with the lengthening of the RNA. Nevertheless, throughout the analysis of phenotype induced in this study, the inhibition of motility precipitated by the 227 bp Gp-flp-6 construct was always more profound than that of the 316 bp construct (i.e., the shorter length of fragment appears to have a superior efficacy). Whether this is a feature specific to Gp-flp-6 or common to other Gp-flps is not known, but all the genes examined could be effectively silenced using dsRNAs of 220–230 bp. These two features of flp gene silencing—concentration and size dependency—mirror the properties of a recently identified molecule critical to RNAi. SID-1 is a putative transmembrane channel that facilitates soaking RNAi by allowing the diffusion of exogenous dsRNA through the channel pore and its systemic spread between cells (43 44 45) . In SID-1-expressing cells, silencing mediated by longer dsRNA constructs was more potent than that elicited by shorter constructs. However, SID-1 does not entirely explain RNAi signal spread, as a recent study recorded low-level spread of an RNAi signal from nerves to muscles in SID-1 mutant C. elegans (30) , and there is growing evidence for the involvement of an endocytic pathway in dsRNA transport between cells (46) . SID-1 is expressed in nematode neurones with externally exposed axons such as those present in sensory structures, as well as in cells exposed to the environment (43) . Thus, there is an overlap between this dsRNA uptake channel distribution and our findings that octopamine is not required for flp gene silencing in G. pallida, suggesting an alternative, nonoral route of RNA uptake. Clearly, dsRNA uptake via the amphids and/or the excretory/secretory pore could serve as a mechanism for exogenous dsRNA to trigger RNAi effects within the phytoparasitic nematode nervous system. In the current study, dsRNA soaking times of >18 h proved critical to the generation of observable phenotypes, with a dramatic reduction in efficacy with soak times ≤12 h. This observation could explain why some authors did not see any RNAi effects after soaking plant parasitic nematodes in various dsRNAs for shorter periods. It seems likely that intracellular dsRNA levels must reach a critical level before the systemic silencing response is triggered and that this requires a considerable period of exposure to the dsRNA trigger.

A summary of the FLPs encoded on the flp genes under investigation here and represented on ESTs for Globodera species may be found in Table 5 . Reflecting the diversity of peptides encoded on flp genes are the diverse and potent effects these neurotransmitters or neuromodulators have on nematode neuromusculature (for reviews, see refs. 11 , 47 , 48 ). The fact that each flp gene silenced here disrupted normal motor capabilities is not surprising in light of the known expression or tissue/cell level activity of the encoded neuropeptides (21) . For instance, in situ hybridization data indicate that Gp-flp-12, Gp-flp-14, and Gp-flp-18 are expressed in diverse motor neurones within G. pallida such that silencing could be expected to compromise normal locomotory capabilities. Similarly, Gp-flp-1 is expressed in interneurones and sensory neurons, where the disruption of normal expression through RNAi could also result in aberrant migratory abilities. Indeed, Gp-flp-1 silenced G. pallida and flp-1 knockout C. elegans (19) both display motor dysfunction, although the detailed phenotypic descriptions appear to be quite different. Expression data for Gp-flp-6 did not firmly establish a role for this gene in motor function, although its expression in interneurones leaves this as a distinct possibility. The Gp-flp-6 encoded peptide KSAYMRFamide is one of the most studied nematode FLPs; it has been identified in representative species throughout the phylum (49 50 51 52) and has been characterized at the gene level from several plant parasitic species (18 ; unpublished observations). Although no obvious phenotype was reported in flp-6 knockout C. elegans, KSAYMRFamide is potently myoactive, eliciting reversible, nerve cord-dependent effects on somatic body wall muscle strips from the parasitic nematode of pigs, Ascaris suum (53) . The only gene silenced here for which there are no expression data is Gp-flp-18, and peptides encoded on this gene have potent effects on a diverse range of tissues from parasitic nematodes (11 , 47 , 48 , 54) . Therefore, none of the phenotypes observed in this study are inconsistent with the gene expression data or available peptide physiology data.


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  		Table 5. FMRFamide-like peptides (FLPs) encoded by Globodera flp genesa

FLPs can induce their effects at remarkably low concentration, and this may explain the transient nature of the RNAi effects we observed with Gp-flp silencing. After a recovery period of 6 days, approximately one-third of the worms that had reduced migratory abilities had returned to normal activity and the suppressive effect had worn off. It seems likely that only a small amount of neuropeptide would be needed to fulfill motor function requirements through its release at selected synapses in the worm. Consequently, it seems plausible that neuropeptide levels would recover quickly after the intervention of RNAi.

This potent modulation of worm neuromusculature suggests that FLP-mediated nematode neurotransmission is important in maintaining normal worm neuromuscular activity. However, it would be premature to assume that modulation of nematode tissues by FLPs in vitro correlates with the role of endogenous FLPs in vivo. In C. elegans, at least 72 FLPs and ~200 other putative neuropeptides have been identified from genome sequence information (14 , 55 , 56) ; however, only ~50 putative neuropeptide receptor genes appear to be present (13 , 57) . This imbalance could imply ligand degeneracy, that is, multiple neuropeptides (e.g., FLPs) interacting with the same receptor. Therefore, the aforementioned responses of nematode muscle preparations to exogenous peptide may not reflect the peptide’s endogenous function; rather, the response may be due to the peptide being applied to, and activating, a receptor in vitro it would never interact with in vivo.

Here, we disrupted G. pallida flp genes in vivo, giving the first indication of the function of the encoded FLPs. The silencing of all those flp genes examined was discernable as a gross inhibition of normal migratory behavior in the migration assay. This inhibition of motility may be attributable to a number of factors. First, it may be a result of dysfunction in body wall muscle coordination manifesting itself as impaired movement to the extent of paralysis. This seems plausible based on the myomodulatory properties of these peptides on nematodes. Conversely, the aberrant phenotype may reflect sensory dysfunction, rendering nematodes incapable of the appropriate orientation responses. A combination of these effects is another possibility. Even without evaluation using the functional assay, disrupted behavioral phenotypes were observed upon silencing each gene of interest, confirming motor dysfunction. Also, some worms that appeared phenotypically normal after gene suppression exhibited nonmigration in the sand column assay (e.g., Gp- flp-12), suggesting the involvement of sensory dysfunction. As stated earlier, most of the flp genes examined here are expressed in motor, sensory, and interneurones, hence their silencing results in pronounced motor and sensory defects. This confirms that neurotransmission/neuromodulation by the FLPs they encode is critical to the efficient control of nematode motor responses. Most important, the silencing of each gene individually resulted in remarkably aberrant phenotypes, even those genes that encode a single peptide. These observations would suggest that redundancy is not a significant component of the FLPergic system, or at least not in the five genes under investigation here. While this does not necessarily mean that multiple peptides cannot or do not activate a single receptor or gate a single channel, it does confirm that each flp gene encodes an integral part of the neuronal circuitry controlling movement through coordination of body wall muscle and/or sensory perception. The remarkable conservation of flp genes across the phylum Nematoda (16) is consistent with functional significance for each flp gene and limited, if any, redundancy.

These data contrast significantly with observations in C. elegans. Although C. elegans flp-1 knockouts display sensory and motor dysfunction (19) , flp-6 and flp-12 knockouts did not display any obvious phenotype; flp-14 and flp-18 knockouts have not been reported. Indeed, although a range of aberrant behaviors has been attributed to flp knockouts, most of the 11 flp mutants examined do not display profound behavioral phenotypes (14 , 56) . Although it is possible that the migration assay used here could place higher demands on G. pallida motor and sensory capabilities than the behavioral assays used to examine C. elegans knockouts, thereby exposing more subtle phenotypes, the profound behavioral phenotypes seen in this study strongly suggest that there are interspecies functional differences in selected FLPs. This observation fits with the reported differences in expression patterns for homologous flp genes in G. pallida and C. elegans (21) .

The ease of application of RNAi to the silencing of neuronal genes in PCN could open the door to unraveling the underlying biology of neuronal systems in these parasitic nematodes. The potent modulation of worm behavior supports the notion that FLP-mediated neurotransmission is critical to nematode biology and also suggests that the FLPergic neuromuscular system may provide attractive targets in parasite control. Exploitation of FLP neuromuscular transmission to control PCN and other parasitic nematodes is an attractive proposition, but will rely on the success of evolving mechanisms to translate in vitro observations to in vivo control measures.


   ACKNOWLEDGMENTS
 
We thank Brendan Mooreland for technical support. We also thank the Department of Agriculture and Rural Development for Northern Ireland for postgraduate studentships for M.J.K., S.M.K, and S.M.M.

Received for publication September 14, 2006. Accepted for publication November 9, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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