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Journal of Nematology 22(4):481-488. 1990. © The Society of Nematologists 1990. Plant-parasitic Nematode Inhibition by and Nematicides 1

C. H. OPPERMAN AND S. CHANG':'

Abstract: The sensitivity of (ACHE) isolated from the plant-parasitic nema- todes Meloidogyne arenaria, M. incognita, and Heterodera glycines and the free-living nematode Cae- norhabditis elegans to carbamate and organophosphate nematicides was examined. The AChE from plant-parasitic nematode species were more sensitive to carbamate inhibitors than was AChE from C. elegans, but response to the was approximately equivalent. The -con- taining phosphate nematicides were poor inhibitors of nematode acetylcholinesterase, but treatment with an oxidizing agent greatly improved inhibition. Behavioral bioassays with living nematodes revealed a poor relationship between enzyme inhibition and expression of symptoms in live nema- todes. Key words: acetylcholinesterase, Caenorhabditis elegans, carbamate, Heterodera glycines, Meloidogyne arenaria, M. incognita, nematicide, organophosphate.

Nematode locomotion depends upon an ilarity in nervous system structure among array of approximately 90 motor neurons diverse groups of nematodes (16), this and interneurons that employ the neuro- scenario probably holds true for plant- transmitters (excitatory) and parasitic nematodes as well. gamma-aminobutyric acid (GABA) (inhib- Carbamate and OP nematicide mode-of- itory) (9,16,17). Acetylcholine causes an in- action is presumed to be inhibition of AChE crease in the rhythmic frequency of muscle (22,26). The presence of AChE in plant- cell depolarizations. The action of acetyl- parasitic nematodes has been demonstrat- at the neuromuscular synapse is ed (1,12,20,21,24,27,29,31,32,36), but few terminated by acetylcholinesterase (ACHE, investigations concerning its physiological E.C. 3.1.1.7). The effects of acetylcholine function have been performed. Aphelen- can be potentiated by numerous AChE in- choides ritzemabosi did not be- hibitors, including carbamate and organo- have as a true acetylcholinesterase (33) but phosphate (OP) nematicides (8,16,30). rather had a broader substrate affinity than Genetic mosaic analysis of Caenorhabditis mammalian forms of the enzyme. This elegans has demonstrated that the only vital finding is supported by data generated with function of AChE in nematodes is to hy- C. elegans (15). drolyze acetylcholine at the neuromuscu- There are several conflicting reports lar synapse (19). Mutant strains that lack concerning nematode sensitivity to AChE AChE in neurons behave as the wild type, inhibitors (2,20,24,28). AChE isolated from provided AChE is present in the muscu- Aphetenchus avenae was sensitive to AChE lature. Those nematodes lacking AChE in inhibitors in vitro, but the compounds the muscles are nonviable. Given the sim- tested were ineffective at comparable levels on A. avenae in bioassays (28). However, A. avenae was extremely sensitive to AChE in- Received for Publication 1 August 1989. i Paper No, 12304 of the Journal Series of the North Car- hibitors at low dosages in a different bioas- olina Agricultural Research Service, Raleigh, NC 27695-7643. say (2). Laboratory tests with other nema- Use of trade names in this publication does not imply en- dorsement by the North Carolina Agricultural Research ser- tode species also have shown nematodes to vice of the products named nor criticism of similar ones not be extremely sensitive to AChE inhibition mentioned. Assistant Professor and Research Technician, Depart- (5). ment of Plant Pathology, North Carolina State University, We have studied AChE in Meloidogyne Raleigh, NC 27695-7616. We thank the Caenorhabditis Genetics Center, which is arenaria, M. incognita, and Heterodera glyci- supported by Contract NOI-AG-9-2113 between the NIH nes as part of a project to examine nervous and the Curators of the University of Missouri, for some of the nematode strains. We also thank Carl D. Johnson and system function in plant-parasitic nema- Stephen P. Schmidt for invaluable counsel and assistance. todes. We used C. elegans as a model be- 481 482 Journal of Nematology, Volume 22, No. 4, October 1990 cause its AChE is very well characterized were aliquoted and stored as decribed (7,15,18,19,23). In this paper we report above. the development of methods to analyze Nematode preparation: In initial experi- plant-nematode enzyme activity, compar- ments, fresh suspensions of packed nema- ative effects of carbamate and OP nema- todes were homogenized by dropwise ad- ticides on isolated nematode AChE com- dition to liquid nitrogen in a chilled mortar pared with living nematodes, and and the liquid nitrogen was allowed to boil experiments addressing bioactivation of away. The remaining frozen beads were parent nematicides to their toxic metabo- crushed to powder with a chilled pestle. lites. The preparation was thawed and then son- icated with a Virtis 50 ultrasonic cell dis- rupter (Gardner, NY) at 50% power for MATERIALS AND METHODS three 5-second bursts. The resulting ho- Nematode culture: Meloido~,ne arenaria mogenate was used immediately for en- and M. incognita were maintained on roots zyme assays and protein determinations (3). of tomato (Lycopersicon esculentum cv. Rut- This method proved to be an inefficient gers) in the greenhouse. Egg masses were use of tissue; therefore, most experiments dissolved with 0.5% sodium hypochlorite utilized a microassay. (13) and the eggs were collected for hatch- Samples (ca. 100 nematodes) were pre- ing. Heterodera glycines was cultured on soy- pared for AChE microassay by subjecting bean (Glycine max cv. Ransom) in the green- them to six freeze-thaw cycles. After the house. Mature cysts were collected by final cycle, samples were incubated at room sieving and gently cracked in a glass ho- temperature overnight. This treatment ef- mogenizer to release the eggs. The eggs fectively disrupts nematode cuticle per- were placed on 25-/zm-pore screens in meability without grossly disrupting the water, and hatched second-stage juveniles morphology and makes the AChE within were collected at 24-hour intervals. The the nematode accessible to substrate. collected nematodes were separated from AChE activity assay: AChE assays were debris by flotation on 20-40% sucrose and carried out using a modification of the sin- rinsed to remove residual sucrose. The gle vial radiometric method (14). Test nematodes were pelleted by centrifugation compounds were solubilized in (718 g, 5 minutes) and then resuspended such that the solvent concentration in the in 1-2 volumes of 100 mM Na borate, 1 reaction mixture did not exceed 2.5%. The mM NaN3 (as a preservative), 1 mg/ml BSA AChE inhibitors tested included the nat- (to prevent denaturative losses), pH 8.0. ural eserine and Packed nematodes were bulk homoge- (Sigma Chemicals, St. Louis, MO); the syn- nized in 1-ml fractions and used immedi- thetic carbamates , , and ately, or aliquots of this suspension con- ; the thiophosphoryl phosphates taining approximately 100 nematodes were , , and ; the stored in 1.5-ml tubes at -80 C for mi- phosphorothioate ethoprop; the phos- croassay. phoramide ; and the phos- Caenorhabditis elegans was cultured on phoryl phosphate (Chem Ser- Escherichia coli 'OP50', on nematode growth vice, West Chester, PA). Nematode medium (NGM) (4). Nematode egg sus- homogenates (40 tzl) (ca. 1.5-2.0 mg/ml pensions prepared by a hypochlorite meth- protein for C. elegans; ca. 0.5-1.0 mg/ml od (11) were added to the plate and incu- for all plant-parasitic nematode species) or bated at 20 C for 4-5 days. Nematodes freeze-thawed nematodes (ca. 100 nema- were harvested by rinsing the plates with todes per tube) were preincubated for 10 borate buffer and were separated from de- minutes with 2 td inhibitor before addition bris and bacteria by flotation on 20-40% of substrate. Final inhibitor concentrations sucrose. Caenorhabditis elegans suspensions in the reaction mixture ranged from 10 -~ Nematode Acetylcholinesterases: Opperman, Chang 483

M to 10 -8 M. Bioactivation of OP inhibi- havior was observed with a 40 x dissecting tors was performed with meta-chlorper- microscope daily for 4-7 days. Controls benzoic acid (mCPBA) (10,34). Inhibitor both with and without solvent were in- and mCPBA were preincubated with the cluded in each assay to determine baseline nematode preparation for 60 minutes in 1 responses and to insure lack of solvent ef- x 10 -4 M concentrations before addition fects on nematode behavior. Results are a of the substrate, 22.9 ~tM [SH]acetylcholine summation of observations from two to ten (TRA-244, Amersham, Arlington Heights, separate repetitions of the assays, with six IL). After a 20-60-minute incubation pe- replications per treatment for C. elegans riod for C. elegans, or 60-360 minutes for and four replications per treatment with the plant-parasitic nematode species, the M. incognita. reaction was terminated by addition of 20 ~tl stop buffer (1 M trichloroacetic acid, 0.5 RESULTS M NaOH, 2 M NaC1) and then 1.3 ml of Incubation of nematode homogenates a -n-butanol scintillation cocktail with [3H]acetylcholine showed a linear in- (0.5% PPO, 0.03% POPOP in toluene). crease in substrate hydrolysis between 10 Substitution of n-butanol for isoamyl al- and 60 minutes. Fractional conversion rates cohol still allows two phasing to occur and of 0.023 + 0.004 u/minute (C. elegans) and is much more economical (15,23). All sam- 0.004 + 0.001 (M. incognita)were obtained ples were counted in a Packard Tri-Carb for uninhibited ACHE. Conversion rates 3255 liquid scintillation spectrometer. comparable to these were achieved using Fractional conversion rates to hydrolyzed 100 freeze-thawed nematodes; therefore, [SH]acetate were calculated to correct for the remainder of these experiments were substrate depletion during the course of performed using this method. Comparison the assay, using the formula: fc units = -In of fractional conversions between fresh ho- (1 - ((nematode CPM - blank)/(total mogenates and freeze-thaw preparations

CPM - blank))). Blank assays contained showed no adverse effect of freeze-thaw no nematode preparation. Total substrate cycles on AChE activity. Incubation of hydrolysis was determined by addition of freeze-thawed C. elegans for 10 minutes 500 U/ml electric eel AChE (Sigma) at the provided a conversion rate of 40-50%, but beginning of the incubation period. There a 3-6-hour incubation was necessary to were two to four separate repetitions of achieve comparable conversion rates for each experiment with three replications per M. arenaria, M. incognita, and H. glycines. treatment. On the basis of saturation curves, prein- Bioassays: Behavioral bioassays were con- cubation with C. elegans homogenate for ducted by incorporating appropriate con- 10 minutes was chosen for experiments in- centrations of test compounds into auto- volving inhibitors. Similar responses were claved NGM (C. elegans) or 1.0% water agar observed for plant-parasitic nematodes. (M. incognita) at 45 C in a water bath. For Kinetic parameters (Km, Vmax) were de- assays involving C. elegans, E. coli OP50 was termined to identify optimum substrate streaked in the middle of the plates and concentrations (data not shown). incubated overnight. For the M. incognita In general, plant-parasitic nematode bioassay, a newly germinated Rutgers to- AChE was more sensitive to carbamate mato seedling was surface sterilized with AChE inhibitors than was C. elegans AChE sodium hypochlorite, placed at one end of (Table 1). This differential response was the plate, and allowed to grow for 48 hours. particularly evident for M. arenaria and M. Nematodes were then placed on the plates incognita where I50 values were up to 65- approximately 5 cm from the root, and the fold lower. In all nematodes, eserine and plates were incubated at 25 C in the dark. neostigmine were the most potent AChE Root penetration was monitored by stain- inhibitors, followed by carbofuran, with al- ing selected roots with acid fuchsin (6). Be- dicarb as the least potent carbamate inhib- 484 Journal of Nematology, Volume 22, No. 4, October 1990

TABLE 1. Concentration of carbamates and organophosphate compounds that reduce nematode acetyl cholinesterase activity by 50% relative to untreated preparations of Caenorhabditis elegans, Heterodera glycines, Meloidogyne arenaria, and M. incognita, t

I~o' molar* Compound C. elegans M. arenaria M. incognita H. glycines

Carbamates Aldicarb 8.5 × 10 -5 5.5 x 10 -6 7.7 × 10 -6 3.1 × 10 -5 Carbofuran 2.3 × 10 -6 9.2 x 10 -8 7.8 × 10 -s 5.1 × 10 -7 Oxamyl 4.9 × 10 -5 7.3 × 10 -7 7.6 × 10 -7 2.1 × 10 -~ Neostigmine 6.5 x 10 -8 2.0 x 10 -9 3,0 x 10 -o 1.0 × 10 -8 Eserine 5.5 x 10 -8 1.0 x 10 -9 1,0 × 10 -9 5.0 × 10 -8

Organophosphates Fenamiphos 6.9 x 10 -5 1.6 x 10 -5 1.0 x 10 -5 3.5 x 10 -5 Ethoprop > 1.0 x 10 -4 > 1.0 x 10 -4 > 1.0 x I0 -~ > 1.0 x 10 -4 Parathion > 1.0 x 10 -4 > 1.0 x 10 -4 > 1.0 x 10 -4 > 1.0 x 10 -4 Paraoxon 7.0 x 10 -7 7.5 × 10 -8 6.0 × 10 -~ 4.0 x 10 -7 Phorate > 1.0 x 10 -4 > 1.0 × 10 -4 > 1.0 x 10 -4 > 1.0 x 10 -4 Terbufos > 1.0 x 10 -4 > 1.0 x 10 -4 > 1.0 × 10 -4 > 1.0 x 10 -4

t AChE activity was measured as hydrolysis of [SH]acetylcholine by freeze-thawed nematode preparations. Percentage of inhibition was calculated as 1-[(treated-blank)/(untreated-blank)] x 100. Approximate conversion rates for untreated nematode preparations were as follows: C. elegans 50%, 20-minute incubation; M. incognita and M. arenaria 35%, 6-hour incubation; H. glycines 6%, 6-hour incubation. Total substrate CPM = 20,000. itor of AChE in vitro for all four nematode ed in an initial period of hyperactivity fol- species. lowed by contracted paralysis. By 24 hours With the exception of fenamiphos and after treatment, nematode muscle tone was paraoxon, the phosphates were very weak completely lost, resulting in flaccid paral- inhibitors of nematode AChE (Table 1). ysis. Occasional twitching and pharyngeal The relative potency of the phosphates on pumping occurred in some nematodes. Ef- AChE from all four nematode species was fects became manifest slightly later in M. similar, with paraoxon the most potent. incognita than in C.elegans, but the pattern AChE from M. arenaria and M. incognita of symptom expression and the minimum were approximately 10,000-fold tess sen- effective concentrations for motility inhi- sitive to fenamiphos than they were to the bition were virtually identical. Carbamates most potent carbamates, and H, glycines possessing a quaternary nitrogen group AChE was approximately 1,000-fold less (eserine and neostigmine) were only slight- sensitive. The phosphates with a sulfur ly inhibitory, and thiophosphoryl phos- moiety in the phosphoryl head (parathion, phates had no effect. Behavioral disruption ethoprop, phorate, and terbufos) exhibit- of M. incognita without coincident paralysis ed poor activity, whereas the phosphoryl was observed at much lower dosages than phosphate (paraoxon) and the phosphoram- were necessary for motility inhibition (Ta- ide (fenamiphos) had activities comparable to ble 2). Although the nematodes appeared the carbamate inhibitors. Dosage-response to move normally, they were unable to pen- curves for aldicarb and fenamiphos inhi- etrate host roots. bition of phytonematode AChE were sim- The poor in vitro activity of OP com- ilar (Fig. 1). The AChE from H. glycines pounds with sulfur in the phosphate head was less sensitive than AChE from M. ar- portiori of the molecule led us to perform enaria and M. incognita to both aldicarb and biological activation experiments with fenamiphos. mCPBA, an oxidant. The greatest gain in Caenorhabiditis elegans and M. incognita activity was seen with parathion oxidized behaved similarly in response to both car- to the phosphate form (paraoxon) (Fig. 2). bamate and OP AChE inhibitors (Table 2). The activation step resulted in a 70% gain Treatment with active compounds result- in potency on H. glycines and smaller in- Nematode Acetylcholinesterases: Opperman, Chang 485

creases on C. elegans, M. arenaria, and M. tO0 incognita (Fig. 2). A concomitant gain in BO H. arenaria ~) activity occurred upon pretreatment of -~ aldtcarb phosphate nematicides with mCPBA (Ta- 60 //~,.,.."/"- ble 3). The dithioates did not gain as much 40 potency by oxidation. Terbufos remained a weak inhibitor on all four nematode 20

ACHE, and phorate moderately gained ac- >'- 0 tivity on AChE from M. arenaria. iQ -8 t0 -7 10 -6 t0 -5 t0-4

¢...3~- t00 DISCUSSION LU 80 14. incognita/. Nematode species exhibit considerable "I" diversity in sensitivity of their AChE to car- "< 6O 1.4_ bamate and OP inhibitors (15,22,23,25,28). C:~ 40 Our data indicate that, with respect to Z AChE inhibition, M. arenaria and M. incog- H 2.0 nita are among the most carbamate-sensi- rn 0 "I" rive nematode species thus far examined. Z 10 -B t0 -7 t0 -6 10-5 t0-4 Paradoxically, they appear to be far less sensitive to paraoxon than expected, based ~e tO0 on their apparent sensitivity to carbamate 80 H. glycJnes /~_ AChE inhibitors (26). Heterodera glycines /J9 showed a similar pattern of relative AChE inhibition by the various inhibitors but was 40 , ~ less sensitive than M. arenaria and M. in- 20 ,// cognita. Generally, differences among the plant-parasitic nematode species in sensi- 0 tivity to synthetic AChE inhibitors were t0-8 t0-7 t0-6 10-5 10-4 small, but considerable variation in in vitro response to the natural earbamates eserine INHIBITOR CONCENTRATION (molar) and neostigmine was observed. The inter- FIG. 1. Effectofvariousconcentrationsofaldicarb and fenamiphos on [3H]acetylcholinehydrolysis by action of the quaternary nitrogen group acetylcholinesterasefrom Meloidogyne arenaria, M. in- present on these compounds with nema- cognita, and Heterodera glycines. Data are means of at tode enzyme active sites may indicate dif- least two experiments using freeze-thaw nematode ferences in AChE conformation among the preparations. species examined. The lack of a clear relationship between ables the soluble AChE and only slowly in- in vitro enzyme toxicity and in vivo data hibits the inaccessible portion of the en- may be related to sublethal behavioral ef- zyme. We have observed behavioral fects (21,28,35). Nematode AChE consists disruption by AChE inhibitors at doses 30- of at least two components in nematodes: fold lower than necessary to inhibit the free a behaviorally relevant component occur- portion of the enzyme. These dosages ring at neuromuscular junctions and freely clearly could not be inhibiting the hidden accessible to inhibitors, and the so-called AChE component. behaviorally irrelevant component, which Inhibition of AChE is a stoichiometric is not readily accessible to inhibitors (30). reaction; i.e., one molecule of inhibitor Animals treated with sufficient AChE in- binds to a single active site per AChE sub- hibitor doses to induce paralysis still pos- unit. Differences in these active sites be- sess substantial in vitro enzyme activity (30). tween AChE species could explain appar- It appears that the inhibitor quickly dis- ent differences within a given nematode 486 Journal of Nematology, Volume 22, No. 4, October 1990

TABLE 2. Behavior of nematodes upon continuous exposure to carbamate and organophosphate inhibitors of acetylcholinesterase.

C. elegans M. incoffnita Compounds 1 hr 24 hr 96 hr MEC 1 hr 24 hr 96 hr MECt PEN:~

Carbamates Aldicarb P+ P++ P++ 5 ~M SE P++ P++ 5 uM 0.26 #M Carbofuran P+ P++ P++ 20 tiM SE P++ P++ 20 #M 10.00 uM Eserine NE NE P ND NE NE NM, RP, G ND ND Neostigmine NE NE P ND NE NE NM, RP ND ND Oxamyl P+ P+ P+ 20 ~zM SE P++ P++ 20 ~M 2.28 #M Untreated NE NE NE NE NE NE Organophosphates Ethoprop P+ P++ P++ 10~M NE P++ P++ 10uM 2.89#M Paraoxon P++ P++,H P÷+ 3p, M NE P++,H P++ 3#M ND Parathion NE NE NE ND NE SE NM, RP ND ND Phenamiphos P+ P++ P++ 3/zM NE P++ P++ 3 #M 0.10 ~zM Phorate NE SE P ND NE SE NM, RP, G ND ND Terbufos NE NE NE ND NE SE NM, RP, G ND ND Untreated NE NE NE NE NE NE Nematodes were placed on inhibitor-amended agar in the presence of a food source (Escherichia coli for Caenorhabditis elegans, and tomato root for Meloidogyne incognita) to elicit a behavioral response. Behavioral responses'. P ~ some slowing; P+ = marked slowing and contraction; P++ = complete paralysis; NE = no effect; ND = not determined; SE = slight effects on movement; NM = normal movement; RP = root penetration; G = galls developing on tomato roots infected with M. incognita. t Minimum effective concentration necessary for symptom expression. Minimum effective concentration necessary for 50% inhibition of penetration of tomato roots by M. incognita.

species in relative power of inhibitors, but results consistent with those obtained with the organismal data do not support this the synthetic analogs. conclusion. Although AChE inhibition data The natural carbamates eserine and revealed 10-1,000-fold differences among neostigmine possess a quaternary nitrogen inhibitors within and between nematode group. Their difficulty in penetrating the species, inhibitors functioned at similar living nematode cuticle could result in the concentrations on the intact nematodes. lack of biological activity observed. In gen- The nematode species in our experi- eral, the carbamates were powerful inhib- ments apparently lack the appropriate itors of nematode soluble ACHE, as ex- cellular machinery to rapidly oxidize sul- pected because they were designed to mimic fur-containing phosphates. This inability the natural substrate, acetylcholine. The suggests that oxidation of carbamate and major function of AChE inhibitors in plant- organophosphate nematicides does not al- ter their efficacy. Ascaris lumbricoides does TABLE 3. Effect of meta-chlorperbenzoic acid on not metabolize organophosphate AChE in- activity of parathion against Caenorhabditis elegans. hibitors either (22). Treatment of inhibi- tors with an oxidizing agent demonstrated Motile AChE nematodest inhibition:~ that their oxidation products are indeed Treatment (%) (%) toxic to nematodes. The relatively small Control 100 0.00 increases in AChE inhibition for the phos- Parathion 100 l 3.61 phorothioates phorate and terbufos are Parathion plus m-CPBA 0 100.00 similar to corresponding increases due to Paraoxon§ 0 97.00 the synthetically prepared metabolites in m-CPBA I00 0.14 Aphelenchus avenae (28). The activation of ~" Motile at 20 minutes at 3 x 10 -4 M. :~ Assayed at 5 x 10 -4 M. parathion to paraoxon and the sulfoxida- § Chemically synthesized analog of parathion + m-CPBA tion of fenamiphos and ethoprop also yield reaction product. Nematode Acetylcholinesterases: Opperman, Chang 487

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COMPOUND (I x 10-4 M) Fl6. 2. Inhibition of nematode acetylcholinesterase activity before and after bioactivation of organo- phosphates by meta-chlorperbenzoic acid. The inhibitor and the mCPBA were incubated for 1 hour before addition of 11.9 ttM [SH]acetylcholine. Percentage of inhibition was calculated as 1 - {(treated - blank)/ (untreated - blank)} x 100, as measured by hydrolysis of [SH] substrate. Paraoxon was the positive control in these experiments. Inhibition by 2 x 10 -4 M paraoxon alone was 99% for Meloidogyne arenaria and 100% for Caenorhabditis elegans, Heterodera glycines, and M. incognita. parasitic nematodes is neuromuscular LITERATURE CITED paralysis. Our data demonstrate that dif- 1. Batterby, S. 1979. Toxic effects ofaldicarb and ferences between C. elegans and plant-para- its metabolites on second-stage larvae of Heterodera sitic species are relatively minor. This is to schachtii. Nematologica 25:377-384. 2. Batterby, S., G. N. J. Le Patourel, and D. J. be expected because of the evolutionary Wright. 1977. Accumulation and metabolism of al- conservation of nervous system develop- dicarb by the free-living nematodes Aphelenchus ave- ment seen in many animal species. nae and Panagrellus redivivus. Annals o£ Applied Bi- Multiple molecular forms of AChE have ology 86:69-76. 3. Bradford, M. 1976. A rapid and sensitive meth- been isolated from C. elegans, A. lumbri- od for the quantitation of microgram quantities of coides, and Stephanurus denatus (15,23). protein utilizing the principle of protein-dye binding. These classes of AChE differ from each Analytical Biochemistry 72:248-254. other in their biochemical properties, in- 4. Brenner, S. 1974. The genetics of Caenorhab- ditis elegans. Genetics 77:71-94. cluding sensitivity to inhibitors. The dif- 5. Bunt, J. A. 1975. Effect and mode of action of ference between species in sensitivity of to- some systemic nematicides. Mededeling Landbow- tal nematode AChE to inhibitors, which we hogeschool Wageningen 75-10:1-125. have reported here, seem likely to be re- 6. Byrd, D. W.,Jr., T. Kirkpatrick, and K. R. Bark- er. 1983. An improved technique for clearing and lated to differential distribution of AChE staining plant tissues for detection of nematodes. classes between these species. Journal of Nematology 15:142-143. 488 Journal of Nematology, Volume 22, No. 4, October 1990

7. Culotti,J. G., G. Von Ehrenstein, M. R. Culotti, 23. Kolson, D. L., and R. L. Russell. 1985. A novel and R. L. Russell. 1981. A second class of acetylcho- class of acetylcholinesterase, revealed by mutations, linesterase deficient mutants of the nematode Cae- in the nematode Caenorhabditis elegans. Journal of norhabditis elegans. Genetics 97:281-305. Neurogenetics 2:93-110. 8. deBell, J. T. 1965. A long look at neuromus- 24. Le Patourel, G. N.J., and D.J. Wright. 1976. cular junctions in nematodes. Quarterly Review of Some factors affecting the susceptibility of two nema- Biology 40:233-251. tode species to phorate. Biochemistry and 9. del CastiUo, J., W. C. de Mello, and T. Morales. Physiology 6:296-305. 1964. Inhibitory action ofgamma-aminobutyric acid 25. Lee, R. M., and M. R. Hodsden. 1963. Cho- on Ascaris muscle. Experientia 20:141-143. linesterase activity in Haemonchus contortus and its in- 10. Drabek, J., and R. Neumann. 1985. Proinsec- hibition by organophosphorous anthelminthics. Bio- ticides. Pp. 35-86 in D. H. Hutson and T. R. Roberts, chemical Pharmacology 12:1241-1252. eds. . New York: Wiley & Sons. 26. Main, A. R. 1976. Structure and inhibitors of 11. Emmons, S. W., M. R. Klass, and D. Hirsh. . Pp. 269-353 in A. M. Goldburg and 1979. Analysis of the constancy of DNA sequences I. Hanin, eds. Biology of function. New during development and evolution of the nematode York: Raven Press. Caenorhabditis elegans. Proceedings of the National 27. McLeod, R. W., and G. T. Khair. 1975. Effect Academy of Sciences USA 76:1333-1337. of oximecarbamate, organophosphate, and benzimid- 12. Huang, S-P., and S. D. Van Gundy. 1978. Ef- azole nematicides on life cycle stages of root-knot fects of aldicarb and its and on the nematodes, Meloidogyne spp. Annals of Applied Biol- biology of Tylenchulus semipenetrans. Journal of Nema- ogy 79:329-341. tology 10:100-106. 28. Pree, D.J.,J. L. Townshend, and D. E. Archi- 13. 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