Subcellular fractions and the refringent granules of the spermatozoa of Ascaris suum (Nematoda)

Summary Six subcellular fractions were isolated by differential centrifugation of the homogenate of spermatozoa of Ascaris suum. The cellular constituents of pelleted fractions, as identified by electron microscopy, were membranes and membranous organelles (fraction A1), microsomal (A2), cytoplasmic (A3), large refringent granules (B1), small refringent granules (B2) and a detergent-soluble fraction (B3).Polypeptide analysis by SDS-PAGE showed that the 18,400-dalton band, one of the major spermatozoan proteins, is detectable in all of the fractions. However, the cytoplasmic (A1) and refringent-granule (B1) fractions contained the highest level.The isolated refringent granules consisted of 2–6 % lipid while the nonlipid fraction formed an insoluble matrix with a fibrillar network morphology. This fibrillar matrix contained three polypeptides of small molecular weight (7,000–14,000) in addition to the 18,400-dalton polypeptide. These small polypeptides (7,000–14,000 MW) are detectable only in fractions of the refringent granules and are therefore called the refringent-granule proteins (RGP). These RGP are sensitive to tryptic hydrolysis and have solubility properties similar to the protein, ascaridine.Adult Ascaris suum were generously provided by Wilson and Company, Cedar Rapids, Iowa. This study was supported by postdoctoral fellowship 5F32AI05646 from NIH. The assistance of Mr. Douglas Wood is gratefully acknowledged     

Copper-zinc superoxide dismutase fromAscaris suum (Nematoda): Purification and characterization

Copper-zinc superoxide dismutase fromAscaris suum (Nematoda) was purified in a new, more efficient, and faster manner. The process included differential centrifugation, fractionation with ammonium sulfate, and sodium dodecyl sulfate-polyacrylamide electrophoresis, yielding a 340-fold purification (specific activity of 47 units/mg). Optimal storage conditions, optimal pH range, thermostability, molecular weight and ultravioltet-visible absorption spectrum of the enzyme are described, and a new enzymatic model for pharmacological screening is suggested.Abbreviations (SOD) Superoxide dismutase - (EDTA) Ethylenediaminetetraacetic acid - (SDS) Sodium dodecyl sulfate - (NBT) p-nitrotetrazolium blue - (UV) Ultraviolet     

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     

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.     

Amino acid and lipid composition of refringent granules from the ameboid sperm of Ascaris suum (Nematoda)

Transformation of the spermatozoon of Ascaris suum from a spheroidal to an ameboid cell is associated with the formation of a motile pseudopodium and coalescence of the intracellular refringent granules. The pseudopodia of the ameboid spermatozoa contain filaments organized into dense patches, bundles, web-like or lace-like networks, as observed by electron microscopy. The morphology and chemistry of the refringent granules were investigated in subcellular fractions enriched for these structures. Isolated refringent granules were heterogeneous in size measuring from 0.5 X 0.6 to 2.3 X 3.5 microns. Each granule is surrounded by a 110 A thick layer. During fusion, the surfaces of the refringent granules form small extensions resembling micropodia. The process of fusion occurs at many sites on a given granule and simultaneous fusion of several granules was commonly observed. Amino acid analyses of the refringent granule proteins (RGP's) indicated: they are rich in aspartic acid or asparagine (48%), leucine (10%), serine (19%) and aromatic amino acids (11%). Gas-liquid chromatographic analyses of alditol acetate derivatives of monosaccharides released by mild acid hydrolysis showed the predominant sugars to be glucose (7.3 micrograms/mg protein), galactose (9.2 micrograms/mg) and N-acetylglucosamine (5.5 micrograms/mg). Lipid analyses indicated a complex mixture of glycerides, ascarosides and waxes, together with a major component that resembled free fatty acid in mobility on TLC.     

Amino acid and lipid composition of refringent granules from the ameboid sperm of Ascaris suum (Nematoda)

Summary Transformation of the spermatozoon of Ascaris suum from a spheroidal to an ameboid cell is associated with the formation of a motile pseudopodium and coalescence of the intracellular refringent granules. The pseudopodia of the ameboid spermatozoa contain filaments organized into dense patches, bundles, web-like or lace-like networks, as observed by electron microscopy.The morphology and chemistry of the refringent granules were investigated in subcellular fractions enriched for these structures. Isolated refringent granules were heterogeneous in size measuring from 0.5×0.6 to 2.3×3.5 m. Each granule is surrounded by a 110 Å thick layer. During fusion, the surfaces of the refringent granules form small extensions resembling micropodia. The process of fusion occurs at many sites on a given granule and simultanenous fusion of several granules was commonly observed.Amino acid analyses of the refringent granule proteins (RGP's) indicated: they are rich in aspartic acid or asparagine (48%), leucine (10%), serine (19%) and aromatic amino acids (11%). Gas-liquid chromatographic analyses of alditol acetate derivatives of monosaccharides released by mild acid hydrolysis showed the predominant sugars to be glucose (7.3 g/mg protein), galactose (9.2 g/mg) and N-acetylglucosamine (5.5 g/mg). Lipid analyses indicated a complex mixture of glycerides, ascarosides and waxes, together with a major component that resembled free fatty acid in mobility on TLC.     

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.     

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.     

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.     

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.     

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.     

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.     

The Ca-activated chloride channel of Ascaris suum conducts volatile fatty acids produced by anaerobic respiration: A patch-clamp study

Plasma membrane vesicles prepared from the bag region of the somatic muscle cell of the parasite Ascaris suum contain a large conductance, voltage-sensitive, calcium-activated chloride channel. The ability of this channel to conduct a variety of carboxylic acids, a number of which are products of anaerobic respiration, was investigated using the patch-clamp technique and isolated inside-out patches of muscle membrane. The channel has a conductance of 140 pS in symmetrical 140 mm chloride. Replacement of internal chloride with various carboxylic acids (140 mm) caused large hyperpolarizing shifts in the reversal potential. Permeability ratios, relative to chloride, were calculated for each acid. The relationship between permeability ratio and ionic size is inverse and linear predicting a pore diameter of 6.55 Å. This simple relationship was not observed between ionic size and conductance. Calculation of the transition state energy required to transfer a single methyl group from aqueous phase to the binding site afforded a value that was low but favorable, indicating a cationic binding site of low field strength. As the channel is able to open fully at the resting membrane potential of Ascaris and is permeable to fatty acids produced by anaerobic respiration, the possible role of this channel in the removal of metabolic products across the muscle membrane is discussed.This work was financed by the Scientific and Engineering Research Council (S.E.R.C.). M. Valkanov was sponsored by The British Council.     

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.     

Single-channel properties of N- and L-subtypes of acetylcholine receptor in Ascaris suum

We are interested in the properties of the target site of cholinergic anti-nematodal drugs for therapeutic reasons. The target receptors are ligand-gated ion channels that have different subtypes, and each subtype may have a different pharmacology. In a contraction assay using the parasitic nematode Ascaris suum, our laboratory has identified several subtypes, including an N-subtype, preferentially activated by nicotine, and an L-subtype, preferentially activated by levamisole. Here we use patch-clamp recordings to test the hypothesis that the single-channel selectivities of nicotine and levamisole are different. Unitary currents evoked by nicotine in this preparation were characterised for the first time. In some patches, both nicotine and levamisole activated small- and large-conductance channels. In other patches, the agonists activated just one channel amplitude. Discriminant analysis allowed classification of the one-conductance patch channels into the small or large categories, based on sets defined by the two-conductance patch data. The small channels had a conductance of 26.1+/-1.5 pS, n=18 (mean+/-SEM); the large conductance channels had a conductance of 38.8+/-1.2 pS, n=23 (mean+/-SEM). Analysis of amplitude histograms of the two-conductance patches showed that nicotine preferentially activated the small-conductance channels and levamisole preferentially activated the large-conductance channels. Our observations suggest that the N-subtype receptor channel has a conductance of 26 pS channel and the L-subtype receptor channel has a conductance of 39 pS.     

A Cl channel in Ascaris suum selectivity conducts dicarboxylic anion products of glucose fermentation and suggests a role in removal of waste organic anions

The permeability of organic anions (produced from anaerobic fermentation of glucose) through a nonselective membrane Cl channel was examined. Single channel recording techniques were used to study the permeabilities of the anions: oxalate, succinate, oxaloacetate, malate, lactate and pyruvate in Ascaris muscle cell membranes. All of the anions, except malate, were found to be conducted through the channel. The relative permeability of most anions could be predicted from the component structure of the anions. The failure of the channel to conduct malate prevents an energy drain on the cell. These studies further the hypothesis that a Cl channel functions to transport waste organic anions across the cell membrane. This mechanism does not require specific exchange carriers for the anions. Channels with properties like the nonselective anion channels of Ascaris, are suitable for transport of carboxylic anions through cell membranes, down a concentration or potential gradient.This work was financed by the Scientific and Engineering Research Council (S.E.R.C.).     

Oral immunogenicity and protective efficacy in mice of transgenic rice plants producing a vaccine candidate antigen (As16) of Ascaris suum fused with cholera toxin B subunit

Cereal crops such as maize and rice are considered attractive for vaccine production and oral delivery. Here, we evaluated the rice Oryza sativa for production of As16—an antigen protective against the roundworm Ascaris suum. The antigen was produced as a chimeric protein fused with cholera toxin B subunit (CTB), and its expression level in the endosperm reached 50 μg/g seed. Feeding the transgenic (Tg) rice seeds to mice elicited an As16-specific serum antibody response when administered in combination with cholera toxin (CT) as the mucosal adjuvant. Although omitting the adjuvant from the vaccine formulation resulted in failure to develop the specific immune response, subcutaneous booster immunization with bacterially expressed As16 induced the antibody response, indicating priming capability of the Tg rice. Tg rice/CT-fed mice orally administered A. suum eggs had a lower lung worm burden than control mice. This suggests that the rice-delivered antigen functions as a prophylactic edible vaccine for controlling parasitic infection in animals.     

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.     

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.     

The mitochondrial ribosomal RNA genes of the nematodes Caenorhabditis elegans and Ascaris suum: Consensus secondary-structure models and conserved nucleotide sets for phylogenetic analysis

The small- and large-subunit mitochondrial ribosomal RNA genes (mt-s-rRNA and mt-1-rRNA) of the nematode worms Caenorhabditis elegans and Ascaris suum encode the smallest rRNAs so far reported for metazoa. These size reductions correlate with the previously described, smaller, structurally anomalous mt-tRNAs of C. elegans and A. suum. Using primer extension analysis, the 5 end nucleotides of the mt-s-rRNA and mt-1-rRNA genes were determined to be adjacent to the 3 end nucleotides of the tRNA^Glu and tRNA^His genes, respectively. Detailed, consensus secondary-structure models were constructed for the mt-s-rRNA genes and the 3 64% of mt-1-rRNA genes of the two nematodes. The mt-s-rRNA secondary-structure model bears a remarkable resemblance to the previously defined universal core structure of E. coli 16S rRNA: most of the nucleotides that have been classified as variable or semiconserved in the E. coli model appear to have been eliminated from the C. elegans and A. suum sequences. Also, the secondary structure model constructed for the 3 64% of the mt-1-rRNA is similar to the corresponding portion of the previously defined E. coli 23S rRNA core secondary structure. The proposed C. elegans/A. suum mt-s-rRNA and mt-1-rRNA models include all of the secondary-structure element-forming sequences that in E. coli rRNAs contain nucleotides important for A-site and P-site (but not E-site) interactions with tRNAs. Sets of apparently homologous sequences within the mt-s-rRNA and mt-1-rRNA core structures, derived by alignment of the C. elegans and A. suum mt-rRNAs to the corresponding mt-rRNAs of other eukaryotes, and E. coli rRNAs were used in maximum-likelihood analyses. The patterns of divergence of metazoan phyla obtained show considerable agreement with the most prevalent metazoan divergence patterns derived from more classical, morphological, and developmental data.Correspondence to: D.R. Wolstenholme