PHYLOGEOGRAPHY OF BAYLISASCARIS PROCYONIS (RACCOON ROUNDWORM) IN NORTH AMERICA.

Sequences of the mitochondrial cytochrome c oxidase 1 (COI) gene of 115 Baylisascaris procyonis individuals from 13 U.S. states and 1 Canadian province were obtained from 44 raccoon hosts to assess genetic variation and geographic structure. The maximum genetic distance between individuals was low (1.6%), consistent with a single species. Moderate COI haplotype (h = 0.60) and nucleotide (π = 0.0053) diversity were found overall. Low haplotype diversity was found among samples east of the Mississippi River (h = 0.036), suggesting that historical growth and expansion of raccoon populations in this region could be responsible for high parasite gene flow or a selective sweep of B. procyonis mtDNA. There was low genetic structure (average Φst = 0.07) for samples east of the continental divide, but samples from Colorado showed higher diversity and differentiation from midwestern and eastern samples. There was marked genetic structure between samples from east and west of the continental divide, with no haplotypes shared between these regions. There was no significant isolation by distance among any of these geographic samples. The phylogeographic patterns for B. procyonis are similar to genetic results reported for their raccoon definitive hosts. The phylogeographic divergence of B. procyonis from east and west of the continental divide may involve vicariance resulting from Pleistocene glaciation and associated climate variation.

DNA Barcoding as a Diagnostic Tool of a Rare Human Parasitosis: The First Case of Lagochilascaris minor in Quintana Roo, Mexico.

Recently, DNA barcoding based on the mitochondrial gene cytochrome c oxidase subunit 1 (COI) has become a widespread tool to identify animals. Its use with parasites of humans has been limited with some groups of nematodes where the amplification of this gene has been difficult. In this study, we present the first COI barcode sequence of a rare parasite from tropical regions, Lagochilascaris minor, which parasitized a human host from Quintana Roo, southern Yucatán Peninsula, Mexico. Destruction of the mastoid apophysis in the lateral sinus and cerebellar involvement were observed at the site of infection. After a radical mastoidectomy and a treatment with 200 mg oral albendazole for 63 days, the patient completely recovered. Lagochilascaris minor was identified based on the ratio between length of spicules and ejaculatory duct, shape of eggs, and host, as well as comparison with its congeners. The mode of infection is unknown, although it could be after direct exposure to eggs or consumption of uncooked wild meat. Morphology of adults is demonstrated using scanning electron microscopy, and high-quality sequences of COI barcode are presented from amplifications using semi-degenerate primers designed for micro-crustaceans. DNA barcoding proved to be a reliable identification method for L. minor. A comparison of the sequences for this species with 81 ascaridoids obtained from the Barcode of Life Database places it in a unique clade most closely related to Baylisascaris procyonis. Future diagnosis of larval and adult stages of L. minor using DNA barcoding will allow the recognition of its infection parameters, transmission, and precise epidemiology. Reports of lagochilascarosis in the Yucatán Peninsula have been occurred over the last decade, suggesting it is an emerging zoonotic disease in the region.

Cloning and characterization of a novel sigma-like glutathione S-transferase from the giant panda parasitic nematode, Baylisascaris schroederi.

BACKGROUND: Baylisascaris schroederi, an intestinal nematode of the giant panda, is the cause of the often fatal disease, baylisascariasis. Glutathione S-transferases (GSTs) are versatile enzymes that can affect parasite survival and parasite-host interactions and, are therefore, potential targets for the development of diagnostic tests and vaccines. METHODS: In this study, we identified a full-length cDNA that encoded a novel, secretory sigma-like GST (Bsc-GSTσ) from a B. schroederi-omic dataset. Following cloning and sequencing, sequence and structural analyses and comparative modeling were performed using online-bioinformatics and proteomics tools. The recombinant Bsc-GSTσ (rBsc-GSTσ) protein was prokaryotically expressed and then used to detect antigenicity and reactivity using immunoblotting assays. In addition, the native protein in female adult B. schroederi was located via immunofluorescence techniques, while the preliminary ELISA-based serodiagnostic potential of rBsc-GSTσ was assessed in native and infected mouse sera. RESULTS: Bsc-GSTσ contained a 621-bp open reading frame that encoded a polypeptide of 206 amino acids with two typical sigma GST domain profiles, including a GST_N_Sigma_like at the N-terminus and a GST_C_Sigma_like at the C-terminus. The presence of an N-terminal signal sequence indicated that Bsc-GSTσ was a secretory protein. Sequence alignment and phylogenetic analyses showed that Bsc-GSTσ was a nematode-specific member of the Sigma class GSTs and shared the closest genetic distance with its homologue in Ascaris suum. Further comparative structure analyses indicated that Bsc-GSTσ possessed the essential structural motifs (e.g., ßαßαßßα) and the consensus secondary or tertiary structure that is typical for other characterized GSTσs. Immunolocalization revealed strong distributions of native Bsc-GSTσ in the body hypodermis, lateral chords, gut epithelium, gut microvilli, oviduct epithelium, and ovaries of adult female worms, similar to its homologue in A. suum. Building on good immunogenic properties, rBsc-GSTσ-based ELISA exhibited a sensitivity of 79.1% and a specificity of 82.0% to detect anti-B. schroederi IgG antibodies in the sera of experimentally infected mice. CONCLUSION: This study presents a comprehensive demonstration of sequence and structural-based analysis of a new, secretory sigma-like GST from a nematode, and its good serodiagnostic performance suggests that rBsc-GSTσ has the potential to detect B. schroederi and, therefore, could be used to develop an ELISA-based serological test to diagnose baylisascariasis in giant pandas.

Proteolytic activity in Hysterothylacium aduncum (Nematoda: Anisakidae), a fish gastrointestinal parasite of worldwide distribution.

Proteases have a significant role in the life cycle of parasites and the pathogen-host relationship, being regarded as important virulence factors. In the parasitic nematode Hysterothylacium aduncum proteolytic activity was measured during in vitro development from third larval stage (L3) to mature adult, using DQ red casein as a fluorogenic substrate. Proteolytic activity was detected in all the developmental stages studied and at all pH values within the range employed (2.0-7.5). The assay with specific inhibitors permitted the determination of metalloprotease activity, and, to a lesser extent, that of aspartate- and cysteine-protease. Serine-protease activity was the lowest of those studied. In L3 recently collected from the host fish (L3-0 h), the greatest activity was found at an optimum pH of 4.0 and was mainly inhibited by 1,10-phenathroline (metalloprotease inhibitor). This metalloprotease activity in L3-0 h (infective stage) may be related to the invasion of the host tissues by this larva. In the other developmental stages, the greatest protease activity was found at pH 5.5, although at pH 4.0 a lower activity peak was detected. On the other hand, our data show that the proteolytic activity of the nematode varies according to the presence of pepsin (an aspartic-protease) in the culture medium. Thus, at pH 4.0, activity was greater in the absence of pepsin, with increasing aspartic-protease activity. Together with the detection of aspartic-, cysteine- and metallo-protease (enzymes involved in digestion in invertebrates) in all the developmental stages of the parasite taking place in the digestive tract of the host fish, this allows us to suggest that the pepsin in the culture medium mimics the predigestion conditions in the habitat of the worm within the host and that the activity detected may have, amongst others, a digestive function.

CO2-fixing enzymes and phosphoenolpyruvate metabolism in the fish parasite Hysterothylacium aduncum (Ascaridoidea, Anisakidae).

CO2 stimulates the development of many of the intestinal helminths that are able to fix CO2 by means of phosphoenolpyruvate carboxykinase (PEPCK), such as Hysterothylacium aduncum. We determined the activity of CO2-fixing enzymes such as PEPCK and phosphoenolpyruvate carboxylase (PEPC), although no significant activity was detected for pyruvate carboxylase or carboxylating-malic enzyme. The former act on phosphoenolpyruvate (PEP) to yield oxalacetate. In the helminths studied, PEP has a vital role in glucidic metabolism. Consequently, we determined the activity of other enzymes involved in the crossroad of PEP, such as pyruvate kinase (PK), lactate dehydrogenase and malate dehydrogenase. All enzymes detected showed significant variations in activity during the in vitro development of the parasite from the third larval stage to mature adult. Fixing of CO2 by PEPCK decreased during development (from 228 to 115 nmol min(-1) mg(-1) protein), while that by PEPC increased (from 19 to 46 nmol min(-1) mg(-1) protein). This enzyme, which is rare in animals, could play a part in detecting levels of free phosphate, releasing it from PEP when required for processes such as glycogenolysis, glycolysis and adenosine 5'-triphosphate (ATP) synthesis. PK, which showed increasing activity during development up to immature adult (from 56 to 82 nmol min(-1) mg(-1) protein), could act in combination with PEPC to obtain energy in the cytosol (in the form of ATP) and in the mitochondria (possible destination of the pyruvate formed), compensating for the decrease in activity of PEPCK.

PCR assays for detection of Baylisascaris procyonis eggs and larvae.

The objective of this study was to develop polymerase chain reaction (PCR) assays for detection of Baylisascaris procyonis eggs and larvae in fecal, environmental, and tissue samples. We have optimized conventional and real-time PCR assays for B. procyonis using the mitochondrial cytochrome oxidase 2 gene as the target for amplification. The lower limit of detection of the parasite genomic DNA was 10 pg in the conventional PCR and 100 fg in the real-time PCR. In both PCR assays, specific amplification of a 146 bp product was achieved with DNA extracted from a single in vitro hatched B. procyonis larva and also from canine fecal samples spiked with as few as 20 unembryonated B. procyonis eggs per gram of feces. The PCR assays were successfully used for detection of B. procyonis eggs and larvae in fecal, environmental, and tissue samples. No DNA amplification was seen when the genomic DNA of related ascarids (including B. transfuga) and a hookworm was used as template in the PCR; however, amplification was seen with the very closely related B. columnaris.

Contracaecum bioccai n. sp. from the brown pelican Pelecanus occidentalis (L.) in Colombia (Nematoda: Anisakidae): morphology, molecular evidence and its genetic relationship with congeners from fish-eating birds.

Contracaecum bioccai n. sp. is described from the brown pelican Pelecanus occidentalis (L.) in northern Colombia (Totumo Marsh) based on 20 enzyme loci studied using multilocus allozyme electrophoresis. Moreover, genetic relationships between the new taxon and related congeners are presented based on allozyme data-sets and sequence analyses (519 bp) of the mtDNA-cox2 gene. Fixed allele differences were found at some of the allozyme loci analysed in comparison with other Contracaecum spp. from pelicans and cormorants [i.e. the sibling species of the C. rudolphii Hartwich, 1964 complex, C. septentrionale Kreis, 1955, C. micropapillatum (Stossich, 1890), C. microcephalum (Rudolphi, 1809) and C. pelagicum Johnston & Mawson, 1942]. The genetic distance, at the allozyme level, between C. bioccai n. sp. and its congeners ranged from D ( Nei ) = 0.80 versus C. septentrionale to D ( Nei ) = 1.40 versus C. micropapillatum. The genetic distance at the mtDNA cox-2 level ranged, on average, from K-2P = 0.12 versus the C. rudolphii species complex to K-2P = 0.15 versus C. micropapillatum. An overall concordant tree topology, obtained from UPGMA and NJ tree analyses inferred from allozyme data, as well as from MP, UPGMA and NJ inferred from mtDNA-cox2 sequence analysis, showed C. bioccai n. sp. as a separated lineage to the other Contracaecum spp. A concordant result was also obtained by PCA analysis based on both the allozyme and mtDNA cox-2 data-sets. All of the tree topologies, derived from the phylogenetic analysis inferred from both allozymes and mtDNA data-sets, were in substantial agreement and depicted C. bioccai as closely related to the sibling species of the C. rudolphii complex (C. rudolphii A and C. rudolphii B) and C. septentrionale. Morphological analysis and a differential diagnosis based on male specimens of C. bioccai, which had been genetically characterised by both allozyme markers and mtDNA sequences analysis with respect to morphologically related congeners, enabled the detection of differences in a numbers of characters, including spicule length, the morphology of the distal end of the spicule and the distribution patterns of the distal caudal papillae.

Molecular detection of sibling species in anisakid nematodes.

The number of sibling species of anisakid nematodes detected over the last two decades has been increased, fuelled by the use of genetic/molecular methodologies. In the present review, we summarize the biological species discovered within most of the nominal species belonging to the genera Anisakis, Contracaecum and Pseudoterranova by the use of allozyme (20-24 loci studied) and recently confirmed by us using mitochondrial cox-2 gene sequence analysis (mtDNA cox-2). Ecological evidence relating to the distributional range of the genetically detected sibling species and their host preferences, which represent data sets that can be utilized for species delimitation and definition, are summarized.

Lagochilascaris minor third-stage larvae secrete metalloproteases with specificity for fibrinogen and native collagen.

Dissemination of parasitic infections depends on migration through tissues and evasion from both hemostatic processes and immune responses from hosts. Metalloproteases play major roles in these mechanisms of pathogen-host interactions and, thus, are considered drug targets. In this study, we characterized metalloprotease activities in excretory/secretory (ES) products from third stage larvae (L3) of the ascarid Lagochilascaris minor, the causative agent of lagochilascariosis, which demonstrates an impressive migrating capacity across host tissues, including bone. Gel enzymography showed that ES products of L3 display two major gelatinolytic activities. Optimal proteolytic activity was found to occur at neutral/alkaline pH and was associated with two L. minor-secreted metalloproteases of 59 (SM59(Lm)) and 114kDa (SM114(Lm)). We next showed that ES products of L3 were able to hydrolyze fibrinogen and collagen I at neutral pH, but not BSA, in an extensive manner. Furthermore, we demonstrated that ES products of L3 mediate hydrolysis of the triple helical structure of collagen I fibers in mouse mesentery. These results suggest that ES proteases of L3 might facilitate both L. minor migration through host tissues by hydrolyzing collagens of the extracellular matrix and evasion from host hemostatic mechanisms by degrading fibrinogen.

Natural antibodies to nematode biotinyl-enzymes in human sera.

Biotinyl-enzymes are conservative molecules present in helminths, as well as in other animals, bacteria and plants. They have recently been found to be antigenic in mice, and a potential source of cross-reactivity among helminths. This study investigated the presence in human sera of antibodies reactive with biotinyl-enzymes from the nematodes Anisakis simplex, Toxocara canis and Ascaris suum. Biotinyl-enzymes from all these nematodes were recognized by IgG1 antibodies in sera from healthy subjects and from Anisakis-free patients infected with other parasites. Interestingly, IgE antibodies reactive with Anisakis simplex biotinyl-enzymes were present in about one third of the sera from Anisakis-free patients infected with other parasites. Our results also demonstrate that the anti-BE IgG1 and IgE antibodies present in the sera of Anisakis-free subjects are cross-reactive among helminths. We conclude that biotinyl-enzymes from nematodes are recognized by natural human antibodies, although Anisakis biotinyl-enzymes do not seem to be the cause of sensitization. Since sera from the Anisakis-free population also present these antibodies, as-yet unidentified factors (dietary components, intestinal inflammation and/or the presence of parasites) may contribute to the induction of anti-BE antibody background.

Contribution to the knowledge of Hysterothylacium aduncum through electrophoresis of the enzymes glucose phosphate isomerase and phosphoglucomutase.

A total of 163 Hysterothylacium aduncum specimens, obtained from two gadoids and one percid, were studied by electrophoresis of the enzymes glucose phosphate isomerase and phosphoglucomutase. The two loci deviated significantly from the Hardy-Weinberg equilibrium, both when considering all specimens and when distinguishing the hosts. This could suggest that there is no single species in either case.

Expression of a single dimeric membrane-bound acetylcholinesterase in Parascaris equorum.

A single form of cholinesterase was detected in the parasitic nematode Parascaris equorum and purified from a low-salt Triton X-100 extract of whole animals by affinity chromatography on an edrophonium-Sepharose matrix. Based on gel-filtration chromatography, sedimentation analysis and SDS-PAGE, such a cholinesterase is an amphiphilic globular (G2) dimer (125-129 kDa, 6.1 S). It includes some hydrophobic domain other than phosphatidylinositol, which gives autoaggregation, detergent interaction and also anchors the molecule to the cell membrane. The enzyme, probably functional in cholinergic neurotransmission, is an acetylcholinesterase showing a fairly low substrate specificity with thiocholine esters. Electrostatic interactions seem to play a major role in the catalytic activity. Studies with inhibitors gave complete inhibition with 1 mM eserine, low sensitivity for procainamide and for tetra(monoisopropyl)pyrophosphortetramide as well as higher inhibition with edrophonium chloride and 1,5-bis(4allyldimethylammoniumphenyl)-pentan-3-one dibromide. The enzyme also showed excess-substrate inhibition with acetylthiocholine. No cross-hybridization occurred between the gene(s) encoding acetylcholinesterase in P. equorum and ace-1 from the free-living nematode Caenorhabditis elegans. The expression of a single cholinesterase form in P. equorum, unusual in free-living nematodes, could be due to parasitic life adaptation with resulting reduction of locomotor activity.

Thienylimidazo[2,1-b]thiazoles as inhibitors of mitochondrial NADH dehydrogenase.

The synthesis of 6-substituted 5-(thienylvinyl)imidazo[2,1-b]thiazoles and 6-thienylimidazo[2,1-b]thiazoles is reported. These compounds were tested as specific inhibitors of the NADH: ubiquinone (UBQ) reductase activity of NADH dehydrogenase in mitochondrial membranes. The 6-thienylimidazo[2,1-b]thiazoles were more potent in mammalian than in nematode mitochondria and had an average titer of 0.11 mM for 2-methyl-6-(2-thienyl)imidazo[2,1-b]thiazole (10). This compound is noncompetitive with the ubiquinone substrate and interacts with a site which is mutually exclusive with that of rotenone but nonexclusive with that of piericidin and several other inhibitors of NADH dehydrogenase. In the series of 5-(thienylvinyl)imidazothiazoles, the hydrobromide of (E)-6-chloro-5-(2-thienylvinyl)imidazo[2,1-b]thiazole (E-5.HBr) was found to be more potent as an inhibitor of the NADH:UBQ activity (IC50 = 15-17 microM) than the 6-thienylimidazoles such as 10. The inhibitory action of E-5.HBr and its analogs is different from that of compound 10 as indicated by the mutual exclusivity with other inhibitors and the relative inhibition of the activity with various electron acceptors.

Two new members in the Contracaecum osculatum complex (Nematoda, Ascaridoidea) from the Antarctic.

The genetic structure of adults and larvae of Contracaecum osculatum (sensu lato) from the Antarctic is analyzed on the basis of 24 enzyme loci. Significant deviations of genotype frequencies from the Hardy-Weinberg equilibrium were found, even in samples recovered from the same host. These data indicate that two distinct, reproductively isolated species coexist in C. osculatum (sensu lato) samples from the Antarctic. They were provisionally designated C. osculatum D and E, as they do not correspond to any of the three species previously detected in this complex from the Atlantic Arctic Boreal region (C. osculatum A, B and C). An allozyme diagnostic key for the identification of the five members of the C. osculatum complex, at the larval and adult stage and in both sexes, is given. Species D and E were found to be genetically quite variable: average P99 = 84.3, A = 3.3 and He = 0.23. Both showed high values of intraspecific gene flow: Nm = 4.6 and 6.1 respectively; similar values were found for the Arctic-Boreal C. osculatum A, B and C. The most related members of the complex are the Antarctic species E and the Arctic-Boreal species A (DNei = 0.21), while the most differentiated ones are the Arctic-Boreal species B and C (DNei = 0.76). The evolutionary divergence of C. osculatum C started more than 3 million years ago, in a Pliocene refugium (Baltic Sea). As to the other C. osculatum species, their evolutionary divergence took place during Pleistocene, when this complex achieved a bipolar distribution. This process involved two distinct colonizations of the marine Antarctic region by ancestors of the northern hemisphere, about 1.5 and 1 million years ago, giving origin to C. osculatum D and E respectively.

The effect of levamisole and albendazole on some enzymes of Ascaridia galli and Heterakis gallinae.

Ascaridia galli and Heterakis gallinae obtained from the common fowl Gallus gallus were exposed to 10(-2)-10(-5)M levamisole and albendazole; both compounds caused death of the parasites in vitro. The effect of the drugs was investigated on homogenates of the treated worms. Albendazole, at 10(-2)M, inhibited oxaloacetate reduction by 67 and 53% and malate oxidation by 21 and 17% in A. galli and H. gallinae, respectively, whereas 10(-4)M levamisole completely inhibited malate dehydrogenase activity in both directions in the two parasites. Lactate dehydrogenase was not affected significantly by either anthelmintic. Aldolase activity was diminished by 57 and 32% in A. galli and H. gallinae, respectively, with 10(-4)M levamisole. Levamisole at 10(-4)M also inhibited the activity of acid and alkaline phosphomonoesterase and cholinesterase. Albendazole had no significant effect on these enzymes in either parasite. Malate dehydrogenase and cholinesterase activity of the host tissue (intestine and caecum) was also reduced significantly with 10(-2) and 10(-3)M levamisole. These studies indicated a multiple mode of action of levamisole and albendazole.

Biochemical and immunological systematics of some ascaridoid nematodes: genetic divergence between congeners.

Vertical starch gel electrophoresis and trefoil immunodiffusion were used to study the systematics of some ascaridoid nematodes. Within the Ascarididae, the time scale of divergence was too great for intergeneric electrophoretic comparisons. Congeneric electrophoretic comparisons of Baylisascaris procyonis (host--raccoon) versus Baylisascaris transfuga (host--black bear), and Toxocara canis (host--domestic dog) versus Toxocara cati (host--domestic cat) yielded Nei genetic distance coefficients of 1.21 and 1.55, respectively. Estimates of times of divergence made from 1 electrophoretic clock calibration suggest that the Baylisascaris species have not shared a common ancestor for 25 million years (Myr), and that the Toxocara species diverged 33 Myr ago. The Baylisascaris divergence estimate corresponds to host-family divergence estimates based on immunological and paleontological evidence, which suggests that cospeciation has occurred. In contrast to this, Ascaris suum (host--pig) and Ascaris lumbricoides (host--human) have a distance coefficient of 0.09. This indicates that these species diverged comparatively recently and may represent a case of host range expansion. Trefoil immunodiffusion comparisons of ascaridoid albumins yielded reactions of identity for A. suum, A. lumbricoides, Parascaris equorum, B. procyonis, B. transfuga, T. canis, and T. cati. This confirms that these taxa are members of a monophyletic group.

Effect of parbendazole and piperazine adipate on the activity of some enzymes of Ascaridia galli and Heterakis gallinae.

Adult Ascaridia galli and Heterakis gallinae obtained from the fowl (Gallus gallus) were treated in vitro with 10(-2) to 10(-5) M parbendazole and piperazine adipate for 10-60 min at 38 degrees C. Both the compounds at 10(-2) M caused mortality of A. galli and H. gallinae after a maximum of 30 min exposure. The effect of the drugs on the homogenates of the treated worm was investigated. Parbendazole (10(-2) M) inhibited malate oxidation by 68% in A. galli and 62% in H. gallinae. Piperazine adipate (10(-2) M) inhibited malate oxidation by 78% in both parasites. In A. galli oxaloacetate reduction was inhibited by 41 and 26% by 10(-2) M parbendazole and piperazine adipate, respectively; with H. gallinae this inhibition was found to be 39 and 55%, respectively. Aldolase activity in both the parasites was also inhibited by 10(-2) M parbendazole and piperazine adipate. Both compounds caused an inhibition of acid phosphomonoesterase activity, but the activities of lactate dehydrogenase and alkaline phosphomonoesterase were not affected significantly. Parbendazole (10(-2) M) had no significant effect on the cholinesterase activity of these parasites, but piperazine adipate (10(-2) M) caused an inhibition of 96% in A. galli and 93% in H. gallinae. The possible mode of action of the drugs is discussed.

Electrophoretic identification of larvae and adults of Anisakis (Ascaridida:Anisakidae).

The relationships between larvae and adults of Anisakis from the Mediterranean Sea and North-East Atlantic Ocean were analysed by multilocus electrophoresis. The correspondence of type I larvae with the A. simplex complex, including the sibling species A. simplex A and B, and of type II larvae with A. physeteris is confirmed. 19 of the 22 loci studied discriminated between the two larval types. Biochemical keys are given for the electrophoretic identification of A. simplex A, A. simplex B and A. physeteris, at both the larval and adult stages.

Genetic structure of Anisakis physeteris, and its differentiation from the Anisakis simplex complex (Ascaridida: Anisakidae).

The genetic structure of Anisakis physeteris from the Mediterranean Sea has been analysed electrophoretically at 22 enzyme loci. The samples studied, although differing in the life-stage (larvae and adults), and in the host (the fishes Micromesistius poutassou and Trachurus trachurus, and the sperm whale Physeter macrocephalus) were genetically homogeneous. Of these loci 11 (Ldh, Sod, Np, Adk-2, Pgm-1, Est-1, Est-2, Acph-1, Acph-2, Lap-2 and Ca) were found to be monomorphic, while the other 11 (Sdh, Mdh, Idh, 6-Pgdh, G3pdh, Got, Adk-1, Pgm-2, Lap-1, Mpi and Gpi) showed from 2 to 7 alleles. The following values of genetic variability were estimated: He = 0.11, P = 0.50, A = 1.95. Distinct alleles were found between A. physeteris and the A. simplex complex at 19 out of the 22 loci studied, and only few rare alleles were shared at the remaining 3 loci. The genetic divergence between A. physeteris and A. simplex A and B is therefore very high, the values of Nei's index D being 7.384 and 6.443 respectively (I = 0.001 and 0.002). The assignation of A. physeteris and the A. simplex complex to two distinct subgenera, Skrjabinisakis and Anisakis, as proposed by Mosgovoy on a morphological basis, appears to be fully justified according to our genetic data.