Role of cysteine 306 in the catalytic mechanism of Ascaris suum phosphoenolpyruvate carboxykinase

Biochemical and metabolic data lead to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes. The Ascaris suum PEPCK shares considerable homology with PEPCK from avian liver and is a good candidate for mutagenesis studies. The Cys306 substitution by Ser and Ala produced active enzymes and the two mutants are kinetically indistinguishable from each other. This substitution affects the catalytic affinity for the formation of the specific enzyme-nucleotide complex (k(cat)/K(m)) in the forward and reverse reactions. Studies with the substrate analogs 2(')dGDP and 2(')dGTP indicate that Cys306 in A. suum PEPCK is one of the residues important in nucleotide binding and may interact with the 2(')OH group in the ribose ring. Alternatively, mutation of this residue could cause protein changes that interfere with the proper conformation of the nucleotides for optimal catalysis to take place.     

Effects of Disinfectants on Larval Development of Ascaris suum Eggs

The objective of this study was to evaluate the effects of several different commercial disinfectants on the embryogenic development of Ascaris suum eggs. A 1-ml aliquot of each disinfectant was mixed with approximately 40,000 decorticated or intact A. suum eggs in sterile tubes. After each treatment time (at 0.5, 1, 5, 10, 30, and 60 min), disinfectants were washed away, and egg suspensions were incubated at 25˚C in distilled water for development of larvae inside. At 3 weeks of incubation after exposure, ethanol, methanol, and chlorohexidin treatments did not affect the larval development of A. suum eggs, regardless of their concentration and treatment time. Among disinfectants tested in this study, 3% cresol, 0.2% sodium hypochlorite and 0.02% sodium hypochlorite delayed but not inactivated the embryonation of decorticated eggs at 3 weeks of incubation, because at 6 weeks of incubation, undeveloped eggs completed embryonation regardless of exposure time, except for 10% povidone iodine. When the albumin layer of A. suum eggs remained intact, however, even the 10% povidone iodine solution took at least 5 min to reasonably inactivate most eggs, but never completely kill them with even 60 min of exposure. This study demonstrated that the treatment of A. suum eggs with many commercially available disinfectants does not affect the embryonation. Although some disinfectants may delay or stop the embryonation of A. suum eggs, they can hardly kill them completely.     

Effects of kimchi extract and temperature on embryostasis of Ascaris suum eggs

To determine the effects of kimchi extracts at different temperatures on larval development, Ascaris suum eggs were mixed with soluble part of 7 different brands of commercially available kimchi and preserved at either 5℃ or 25℃ for up to 60 days. A. suum eggs incubated at 25℃ showed marked differences in larval development between kimchi extract and control group. While all eggs in the control group completed embryonation by day 21, only 30% of the eggs in the kimchi extract group became embryonated by day 36 and about 25% never became larvated even at day 60. At 5℃, however, none of the eggs showed larval development regardless of the incubation period or type of mixture group. To determine the survival rate of A. suum eggs that showed no embryonation after being preserved at 5℃, eggs preserved in kimchi extracts for 14, 28, and 60 at 5℃ were re-incubated at 25℃ for 3 weeks in distilled water. While all eggs in the control group became larvated, eggs in the kimchi extract group showed differences in their embryonation rates by the incubation period; 87.4 % and 41.7% of the eggs became embryonated after being refrigerated for 14 days and 28 days, respectively. When refrigerated for 60 days, however, no eggs mixed in kimchi extract showed larval development. Our results indicate that embryogenesis of A. suum eggs in kimchi extract was affected by duration of refrigeration, and that all eggs stopped larval development completely in kimchi kept at 5℃ for up to 60 days.     

Ascaris suum: immunoglobulin responses in mice

The immune response in mice to infection with Ascaris suum was characterized by determining (1) changes in serum immunoglobulin levels and (2) changes in the relative proportions of immunoglobulin-containing cells in major lymphoid tissues and sites of possible local immunoglobulin production.     

Effects of Some Pesticides on Development of Ascaris suum Eggs

To evaluate the effects of pesticides to parasite eggs, Ascaris suum eggs were incubated with 5 different pesticides (1:1,500-1:2,000 dilutions of 2% emamectin benzoate, 5% spinetoram, 5% indoxacarb, 1% deltamethrin, and 5% flufenoxuron; all v/v) at 20℃ for 6 weeks, and microscopically evaluated the egg survival and development on a weekly basis. The survival rate of A. suum eggs incubated in normal saline (control eggs) was 90±3% at 6 weeks. However, the survival rates of eggs treated with pesticides were 75-85% at this time, thus significantly lower than the control value. Larval development in control eggs commenced at 3 weeks, and 73±3% of eggs had internal larvae at 6 weeks. Larvae were evident in pesticide-treated eggs at 3-4 weeks, and the proportions of eggs carrying larvae at 6 weeks (36±3%-54±3%) were significantly lower than that of the control group. Thus, pesticides tested at levels similar to those used in agricultural practices exhibited low-level ovicidal activity and delayed embryogenesis of A. suum eggs, although some differences were evident among the tested pesticides.     

Maintenance of Dispersed Reproductive Cells from Male and Female Ascaris suum

In vitro cultivation of tissues and cells provides an experimental methodology to define and manipulate physiological mechanisms that are not possible with in vivo techniques. Tissues from the germinative-growth zones of adult Ascaris suum gonads were excised and minced, and then enzymatically dispersed and transferred to an artificial, perienteric fluid-fetal calf-serum-medium complex. Cells were maintained in a viable state for 8 days, with medium replacement every 48 hours. During this period, morphological changes in the gonadal cells included decreased size, dedifferentiation, and degeneration. Two indices of metabolism, evolution of ¹⁴CO₂ from radiolabelled glucose and reduction of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium), decreased by approximately 50% and 60%, respectively. The in vitro procedures developed provide the first opportunity to examine specific cellular functions of nematode reproductive tissues over an extended period of time.     

The effect of opiates and opiate antagonists on heat latency response in the parasitic nematode Ascaris suum

The effects of the opiates morphine and morphine-6-glucuronide (M6G), the mu opioid receptor specific antagonist D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NH(2) (CTOP), and the general opiate antagonist naloxone on the latency of response to thermal stimulation were determined in the parasitic nematode Ascaris suum. Thermal detection and avoidance behaviors of the worms were evaluated with a tail flick analgesia meter using a modification of a technique employed for nociception experiments in rodents. Morphine and M6G were shown to have a dose dependent analgesic effect on A. suum's latency of response to heat with morphine being the most potent. The analgesic effect of morphine was reversed by naloxone but not CTOP. Neither naloxone nor CTOP was able to block the analgesia of M6G. CTOP but not naloxone had significant analgesic effects on its own. These findings are generally consistent with previous results on the effects of opiates and nitric oxide release from A. suum tissue. Apparently these nematodes possess opioid receptors that effect nociception.     

Vaccination with recombinant Ascaris suum 24-kilodalton antigen induces a Th1/Th2-mixed type immune response and confers high levels of protection against challenged Ascaris suum lung-stage infection in BALB/c mice

Previous studies have shown that antigens from various life-cycle stages of Ascaris suum can induce host-protective immunity against challenge infections with infective eggs of A. suum. This study evaluated whether Escherichia coli-expressed recombinant 24-kDa antigen from A. suum (rAs24) was a suitable vaccine candidate for the control of Ascaris infections by examining its performance in a mouse model. Immunization of BALB/c mice in three consecutive doses with rAs24 in Freund's Complete Adjuvant (FCA) results in protection against challenge infections as manifested by a 58% reduction (P<0.001) in recovery and stunted development of A. suum lung-stage larvae at day 7 post-challenge. Sera obtained from immune protected mice had a significantly increased level of immunoglobulin G (IgG) (P<0.0001) but had no IgE response. Analysis of IgG-subclass profiles revealed that IgG1 (P<0.0001) showed the greatest increase followed by IgG2b (P<0.005), IgG2a (P<0.006) and IgG3 (P<0.04). Splenic T cells from rAs24-FCA immunized mice secreted significantly high levels of both Th1 cytokine gamma-interferon (P<0.005) and Th2 cytokine interleukin-10 (P<0.001) after stimulation with rAs24 in vitro. Interestingly, affinity purified anti-rAs24 IgG was shown to inhibit moulting of A. suum lung-stage L3 to L4 in vitro by 26%, indicating an in vivo function of the endogenous As24 in the moulting processes. An intense expression of endogenous As24 in the hypodermis and gut epithelium of A. suum lung-stage L3 by immunofluorescence supports a function for endogenous As24. These findings may contribute to the understanding of rAs24-induced Th1/Th2-mediated effector mechanisms required for the protection of A. suum lung-stage larval infection.     

Ascaris suum cytochrome b5, an adult-specific secretory protein reducing oxygen-avid ferric hemoglobin

The anaerobic parasitic nematode Ascaris suum has an oxygen-avid hemoglobin in the perienteric fluid, the biological function of which remains elusive. Here, we report that Ascaris cytochrome b₅ is expressed specifically in the intestinal parasitic stage and is secreted into the perienteric fluid, thus co-localizing with Ascaris hemoglobin. We also found that cytochrome b₅ reduces Ascaris non-functioning ferric methemoglobin more efficiently than mammalian methemoglobin. Furthermore, a computer graphics model of the electron transfer complex between Ascaris cytochrome b₅ and Ascaris hemoglobin strongly suggested that these two proteins are physiological redox partners. Nitric oxide has been reported to react easily with oxygen captured in hemoglobin to form nitrate, but not toxic free radicals, which may result in production of methemoglobin for the cytochrome b₅ to regenerate functional ferrous hemoglobin. Therefore, our findings suggest that Ascaris cytochrome b₅ is a key redox partner of Ascaris hemoglobin, which acts as an antioxidant.     

The ovijector of Ascaris suum: multiple response types revealed by Caenorhabditis elegans FMRFamide-related peptides

Caenorhabditis elegans possesses 22 FMRFamide-like peptide (flp) genes predicted to encode 60 different FMRFamide-related peptides with a range of C-terminal signatures. Peptides from five flp genes (1, 6, 8, 9 and 14) are known to modulate the ovijector of Ascaris suum in vitro. This study examines the physiological effects of peptides from the remaining 17 flp genes such that the variety of FMRFamide-related peptide-induced ovijector response types can be delineated. Five categories of response were identified according to the pattern of changes in contractile behaviour and baseline tension. Peptides encoded on 16 flp genes (1, 2, 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 20) had qualitatively similar inhibitory (response type 1) actions, with the lowest activity thresholds (1 nM) recorded for peptides with FIRFamide or FLRFamide C-terminal signatures. Peptides encoded on four flp genes (2, 18, 19 and 21), and on the A. suum afp-1 gene, had excitatory actions on the ovijector (response type 2), with PGVLRFamides having the lowest activity threshold (1 nM). An flp-2 peptide (LRGEPIRFamide) induced a transient contraction of the ovijector (activity threshold, 10nM) that was designated response type 3. Response type 4 comprised a transient contraction followed by an extended period of inactivity and was observed with peptides encoded on flp-5 (AGAKFIRFamide, APKPKFIRFamide), flp-8 (KNEFIRFamide) and flp-22 (SPSAKWMRFamide). SPSAKWMRFamide was the most potent peptide tested with an activity threshold of 0.1 nM. A single peptide (AMRNALVRFamide; activity threshold 0.1 microM), encoded on flp-11, induced response type 5, a shortening of the ovijector coupled with an increase in contraction frequency. Although most flp genes encode structurally related peptides that trigger one of the five ovijector response types, flp-2 and flp-11 co-encode FMRFamide-related peptides that induce distinct responses. Within the ovijector of A. suum FaRPs play a complex role involving at least five receptor subtypes or signalling pathways.     

Effects of SDPNFLRF-amide (PF1) on voltage-activated currents in Ascaris suum muscle

Helminth infections are of significant concern in veterinary and human medicine. The drugs available for chemotherapy are limited in number and the extensive use of these drugs has led to the development of resistance in parasites of animals and humans ( [Geerts and Gryseels, 2000], [Kaplan, 2004] and [Osei-Atweneboana et al., 2007]). The cyclooctadepsipeptide, emodepside, belongs to a new class of anthelmintic that has been released for animal use in recent years. Emodepside has been proposed to mimic the effects of the neuropeptide PF1 on membrane hyperpolarization and membrane conductance (Willson et al., 2003). We investigated the effects of PF1 on voltage-activated currents in Ascaris suum muscle cells. The whole cell voltage-clamp technique was employed to study these currents. Here we report two types of voltage-activated inward calcium currents: transient peak (I_peak) and a steady-state (I_ss). We found that 1 μM PF1 inhibited the two calcium currents. The I_peak decreased from −146 nA to −99 nA (P = 0.0007) and the I_ss decreased from −45 nA to −12 nA (P = 0.002). We also found that PF1 in the presence of calcium increased the voltage-activated outward potassium current (from 521 nA to 628 nA (P = 0.004)). The effect on the potassium current was abolished when calcium was removed and replaced with cobalt; it was also reduced at a higher concentration of PF1 (10 μM). These studies demonstrate a mechanism by which PF1 decreases the excitability of the neuromuscular system by modulating calcium currents in nematodes. PF1 inhibits voltage-activated calcium currents and potentiates the voltage-activated calcium-dependent potassium current. The effect on a calcium-activated-potassium channel appears to be common to both PF1 and emodepside (Guest et al., 2007). It will be of interest to investigate the actions of emodepside on calcium currents to further elucidate the mechanism of action.     

Effect of Temperature on Embryonation of Ascaris suum Eggs in an Environmental Chamber

The influence of temperature on the development and embryonation of Ascaris suum eggs was studied using coarse sand medium in an environmental chamber with 50% humidity. The time required for development and embryonation of eggs was examined under 3 different temperature conditions, 5℃, 25℃, and 35℃. A. suum eggs did not develop over 1 month at the temperature of 5℃. However, other temperature conditions, 25℃ and 35℃, induced egg development to the 8-cell-stage at days 5-6 after incubation. All eggs examined developed to the 8-cell stage at day 6 after incubation in the sand medium at 25℃. The higher temperature, 35℃, slightly accelerated the A. suum egg development compared to 25℃, and the development to the 8-cell stage occurred within day 5 after incubation. The formation of larvae in A. suum eggs at temperatures of 35℃ and 25℃ appeared at days 17 and 19 after incubation, respectively. These findings show that 35℃ condition shortens the time for the development of A. suum eggs to the 8-cell-stage in comparison to 25℃, and suggest the possibility of accelerated transmission of this parasite, resulting from global warming and ecosystem changes.     

Quantitative Evaluation of Viability- and Apoptosis-Related Genes in Ascaris suum Eggs under Different Culture-Temperature Conditions

Ascaris suum eggs are inactivated by composting conditions; however, it is difficult to find functional changes in heat-treated A. suum eggs. Here, unembryonated A. suum eggs were incubated at 20℃, 50℃, and 70℃ in vitro, and the gene expression levels related to viability, such as eukaryotic translation initiation factor 4E (IF4E), phosphofructokinase 1 (PFK1), and thioredoxin 1 (TRX1), and to apoptosis, such as apoptosis-inducing factor 1 (AIF1) and cell death protein 6 (CDP6), were evaluated by real-time quantitative RT-PCR. No prominent morphological alterations were noted in the eggs at 20℃ until day 10. In contrast, the eggs developed rapidly, and embryonated eggs and hatched larvae began to die, starting on day 2 at 50℃ and day 1 at 70℃. At 20℃, IF4E, PFK1, and TRX1 mRNA expression was significantly increased from days 2-4; however, AIF1 and CDP6 mRNA expression was not changed significantly. IF4E, PFK1, and TRX1 mRNA expression was markedly decreased from day 2 at 50℃ and 70℃, whereas AIF1 and CDP6 mRNA expression was significantly increased. The expressions of HSP70 and HSP90 were detected for 9-10 days at 20℃, for 3-5 days at 50℃, and for 2 days at 70℃. Taken together, incremental heat increases were associated with the rapid development of A. suum eggs, decreased expression of genes related to viability, and earlier expression of apoptosis-related genes, and finally these changes of viability- and apoptosis-related genes of A. suum eggs were associated with survival of the eggs under temperature stress.     

AF2 interaction with Ascaris suum body wall muscle membranes involves G-protein activation

KHEYLRF-NH(2) (AF2) is the most abundant FMRFamide-related peptide (FaRP) in Ascaris suum and also in many other parasitic and free-living nematodes. The AF2 abundance in the highly diverse nematodes and its potent and profound effects on the neuromuscular systems make AF2 and its receptor(s) very attractive targets for the discovery of novel broad-spectrum anthelmintics. Although FaRP receptors are believed to belong to the large family of G-protein coupled receptors (GPCRs), to date no AF2 receptor(s) have been cloned so there is no final proof to show that they are indeed G-protein coupled. In this study, using A. suum body wall muscle membranes, we showed that: (1) AF2 effectively (EC(50) 57 nM) induced a dose-dependent stimulation of [35S]GTP gamma S binding to the membranes, which is a hallmark of G-protein activation; (2) the high affinity binding of [125I-Tyr(4)]AF2 was inhibited in a dose-dependent manner by GTP with a K(i) of 10.5 nM (so-called guanine nucleotide effect, characteristic for GPCRs). Collectively, our results provide direct evidence for G-protein involvement in AF2-triggered receptor activation and thus confirm that the receptor for AF2 in A. suum is a GPCR.     

Effects of KHEYLRFamide and KNEFIRFamide on cyclic adenosine monophosphate levels in Ascaris suum somatic muscle

KHEYLRF-NH(2) (AF2) is a FMRFamide-related peptide (FaRP) present in parasitic and free-living nematodes. At concentrations as low as 10 pM, AF2 induces a biphasic tension response, consisting of a transient relaxation followed by profound excitation, in neuromuscular strips prepared from Ascaris suum. In the present study, the effects of AF2 on cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and inositol-1,4,5-triphosphate (IP(3)) levels were measured following muscle tension recordings from 2 cm neuromuscular strips prepared from adult A. suum. AF2 induced a concentration- and time-dependent increase in cAMP, beginning at 1 nM; cAMP levels increased by 84-fold following 1 h exposure to 1 microM AF2. cGMP and IP(3) levels were unaffected by AF2 at concentrations 相似文献   

Microevolutionary Patterns and Molecular Markers: The Genetics of Geographic Variation in Ascaris suum

Molecular markers have been used only rarely to characterize the population genetic structure of nematodes. Published studies have suggested that different taxa may show distinct genetic architectures. Isoenzyme and RAPD markers have been used to investigate geographic variation of Ascaris suum at the level of infrapopulations (nematodes within individual hosts), within localities, and among geographic regions. Independent estimates of genetic differentiation among population samples based on isoenzyme and RAPD data showed similar patterns and substantial correlation. Heterozygote deficiencies within infrapopulations and large values for inbreeding coefficients among infrapopulations suggested that the composition of these populations was not consistent with a model of random recruitment from a large panmictic pool of life-cycle stages. Both isoenzyme and RAPD markers revealed moderate levels of genetic differentiation among samples representing infrapopulations and localities. Of total gene diversity, 9.4% (isoenzyme) and 9.2% (RAPD) was partitioned among infrapopulations. Geographic localities accounted for 7.8% (isoenzyme) and 6.2% (RAPD) of total diversity. Only infrapopulations from the same farm had low levels of differentiation.     

Biophysical characterization of a large conductance anion channel in hypodermal membranes of the gastrointestinal nematode,Ascaris suum

Patch-clamp recordings from muscle- and cuticle-facing hypodermal membranes of the gastrointestinal nematode Ascaris suum reveal a high-conductance, voltage- sensitive Ca(2+) -dependent Cl(-) channel. The hypodermal channel has a conductance of 195 pS in symmetrical 160 mM NaCl. The open probability of the channel is highly voltage-sensitive, and channel activity is not observed when Ca(2+) is reduced to <100 microM. The channel is permeable to organic anions that are major end-products of carbohydrate metabolism in A. suum, including acetate, butyrate and 2-methylvalerate. The conductances and relative permeabilities of these organic anions are inversely related to size, with 2-methylvalerate being only approximately 3% as permeable as Cl(-). The diameter of the channel pore was 12.3+/-0.2 A, calculated from the relative permeability coefficients of Cl(-) and the organic anions. Results of this study are consistent with the hypothesis that the large conductance anion channel in A. suum hypodermal membranes provides a low energy pathway for organic anion excretion from the hypodermal compartment, followed by diffusion across the aqueous channels of the cuticle matrix.     

In vitro activation and behavior of the ameboid sperm of Ascaris suum (Nematoda)

Summary A system is described for the study of activation and motility of Ascaris spermatozoa in vitro. Activation was accomplished by addition of the sperm-activating substances (SAS), extracted from the male accessory gland, to cells incubated in phosphate-buffered saline (pH 7.4) at 37–39° C under anaerobic conditions (95% N₂, 5% CO₂). Activation is characterized by a change from spherical to ameboid shape with coalescence of the refringent granules. The normal ameboid spermatozoa bear several stubby and needle-like filopodia at the lamellipodial margin. Within the lamellipodium are bundles of microfilament-like structures extending toward the pseudopodial membrane and concentrating within the needle-like filopodia. These filopodia exhibit a pendulous, sweeping motion with subsequent retraction and disappearence within the main lamellipodium. Membranes of the ameboid cells interact at the pseudopodial regions with partial fusion, as suggested by apparent membrane breakdown between interdigitating portions of the pseudopodia. Activation is complete in 5–15 min, is totally inhibited at 4° C and/or by an atmospheric environment, but can be reinitiated by transfer to anaerobic conditions at 22–9° C. Activation also requires favorable pH (6.8–8.7) and continual exposure to sufficiently high sodium concentrations (134–154mM), i.e., lowering of sodium concentration to 10 mM causes irreversible inactivation. Sodium may be replaced by potassium or lithium but not by Tris or sucrose. Proteinases (10 g/ml) can act as activators even though SAS lack detectable proteolytic activity against azoalbumin, azocasein, TAME and BTEE and SAS activation was not inhibited by TLCK or soybean trypsin inhibitor.Adult Ascaris suum were provided through the generosity of Wilson and Company, Cedar Rapids, Iowa, U.SA. This study was supported by grant number 5T01 HD00152 and postdoctoral fellowship 1F 32AI05646 from the National Institute of Health, U.S.A.     

Ultrastructural observations on the cell surface of the intestinal epithelium of the nematode,Ascaris suum

Summary The intestinal epithelium of Ascaris suum consists of a single layer of tall columnar epithelial cells that rest on a thick basal membrane in contact with the pseudocoelomic cavity. Experiments were conducted on glutaraldehyde-fixed tissue to ascertain the nature of the electronegative charges associated with both the apical microvillar surface and basal membrane.A strong electronegative charge was demonstrated on the microvillar surface and basal membrane with ruthenium red and cationic ferritin staining. The ionic nature of ferritin binding was demonstrated with poly-L-lysine, a polycation that interacts with anionic groups on the membrane and thus blocks the subsequent binding of ferritin. Tissue thus treated was devoid of reaction product. Methylation with diazomethane completely abolished staining. Since the stronger acidic groups of sulfates or phosphates would not be protonated under the conditions employed in this study, and therefore susceptible to methylation, staining by ferritin is thought to be due to its interaction with carboxyl groups. Prior enzymatic treatment of tissue with neuraminidase or phospholipase C had no effect on subsequent ferritin binding. Tissue exposed to colloidal iron at various pH values showed maximal reactivity at a pH of 2.5 or above. Above pH 2.5, the dissociation of protons from free carboxyl groups of protein-bound amino-acid residues with pK's of 3.8 and 4.2 would be maximal, and the ionized carboxyl groups are then available to interact with iron micelles. These results suggest the presence of weaker acidic groups, such as the carboxyl groups of acidic amino acids or uronic acid residues. The stronger acidic groups of sialic acid and the esterified sulfate groups, if present, contribute only minimally to overall staining. These results demonstrate that a high electronegative charge density exists, despite the apparent lack of sialic acid. Staining is believed to be due to carboxyl groups of acidic amino acids and/or carboxyl groups or uronic acid residues.Part of this work was conducted at the Department of Zoology, Louisiana State University, Baton Rouge, Louisiana     

Mitochondrial DNA Sequence Divergence among Meloidogyne incognita,Romanomermis culicivorax,Ascaris suum,and Caenorhabditis elegans

Mitochondrial DNA sequences were obtained from the NADH dehydrogenase subunit 3 (ND3), large rRNA, and cytochrome b genes from Meloidogyne incognita and Romanomermis culicivorax. Both species show considerable genetic distance within these same genes when compared with Caenorhabditis elegans or Ascaris suum, two species previously analyzed. Caenorhabditis, Ascaris, and Meloidogyne were selected as representatives of three subclasses in the nematode class Secernentea: Rhabditia, Spiruria, and Diplogasteria, respectively. Romanomermis served as a representative out-group of the class Adenophorea. The divergence between the phytoparasitic lineage (represented by Meloidogyne) and the three other species is so great that virtually every variable position in these genes appears to have accumulated multiple mutations, obscuring the phylogenetic information obtainable from these comparisons. The 39 and 42% amino acid similarity between the M. incognita and C. elegans ND3 and cytochrome b coding sequences, respectively, are approximately the same as those of C. elegans-mouse comparisons for the same genes (26 and 44%). This discovery calls into question the feasibility of employing cloned C. elegans probes as reagents to isolate phytoparasitic nematode genes. The genetic distance between the phytoparasitic nematode lineage and C. elegans markedly contrasts with the 79% amino acid similarity between C. elegans and A. suum for the same sequences. The molecular data suggest that Caenorhabditis and Ascaris belong to the same subclass.