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Cystic echinococcosis is a globally distributed zoonotic disease of great medical and veterinary importance, which is caused by the cestode Echinococcus granulosus sensu lato. In Ukraine, two areas of the prominent circulation of the parasite are established, the southern steppe zone with sheep as the main transmitter, and the northern forest-steppe zone and Polissia, where pigs are mainly responsible for maintaining the E. granulosus transmission. Given that only a few studies have so far addressed the genetic diversity of the parasite in Ukraine, we have sequenced partial mitochondrial genes of cytochrome c oxidase 1 (789 bp), NADH dehydrogenase 1 (602 bp) and 12S rRNA (333–334 bp) in pig metacestodes from the Sumy region (farms close to Sumy, northeastern Ukraine) and the Kyiv region (a farm in Bila Tserkva, central Ukraine). Four isolates from four pigs in the Sumy region were identified as E. canadensis (G7 genotype), the major E. granulosus s.l. species circulating in Eastern Europe, including the three microvariants (G7A, G7B, G7C). Three isolates from the two pigs in the Kyiv region were classified as E. granulosus s.s. (G1 genotype), including one microvariant (G1A). To our knowledge, this is the first genetic record of E. granulosus s.s. with the presumed highest infectivity and virulence among the E. granulosus s.l. species in Ukraine. The finding has implications for public health as local control programmes should take into consideration different development rate of this parasite in dogs and the greater risk of the species for human infection.

Whipworms are responsible for up to 500 million cases of trichuriasis worldwide, with higher endemicity in tropical and sub-tropical countries. In non-endemic countries, trichuriasis can be accidentally diagnosed upon colonoscopy, often in the presence of negative microscopy. Here, we describe an incidental diagnosis of trichuriasis in an HIV patient residing in a non-endemic area (i.e., Turin, Italy), six months after his return from Antigua. The species-level diagnosis was made thanks to PCR-based molecular identification of Trichuris sp. following optical microscopy detection. Overall, this case highlights the importance of improving parasitic diseases diagnosis through cutting-edge clinical and laboratory diagnostic tools alongside advanced training of specialists in the area of parasitology.

Monoxenous parasites may easily infect animals in captivity, and nematodes belonging to the genus Trichuris are commonly reported in zoological gardens worldwide. Infections in captive animals should be accurately monitored and the characterization of pathogens is highly advisable, as a tool to infer possible routes of intra- and interspecific transmission pathways and to assess the related zoonotic potential. Whipworms are usually identified on the basis of few morphological features of adults males and eggs and by an host-affiliation criterion. Given the strong morphological convergence of adaptive traits and the possible occurrence of hybridization and/or cross-infections events, the use of molecular methods is of great utility. Here, we analysed two partial mitochondrial loci, the cytochrome c oxidase I and the cytochrome b regions, in Trichuris spp. infecting four animal species hosted in the Bioparco Zoological Garden of Rome. Results from molecular systematics, compared to previous data, suggested that the five Trichuris taxa recovered were well separated, showing a significant degree of host affiliation (herbivorous, primates/swine and rodents/canids). The screw horn antelopes and the camels were infected with two variants of Trichuris ovis; Trichuris sp. similar to those observed in rodents from South America was infecting the Patagonian maras. Moreover, Trichuris from the ring-tailed lemur showed a great similarity to Trichuris infecting the Japanese macaque previously analysed from the same zoological garden, and clustering together with Trichuris trichiura, posing a potential zoonotic threat for visitors and workers.

The European distribution of Echinococcus multilocularis has been reported to be expanding east and north, whereas its southern limits are deemed stable. During a study on Echinococcus granulosus s.l. infections in wolves and dogs in the Italian Maritime Alps, we unexpectedly detected the presence of E. multilocularis eggs in four fecal samples from at least two shepherd dogs, and in five wolf fecal samples. This finding, in an area about 130 km south of the southernmost E. multilocularis report in the Alps, may suggest a rapid expansion southward. While infections in foxes are currently being investigated, these data seem to indicate the potential for a new E. multilocularis endemic area. If this will be confirmed, the implementation of surveillance programs in wild and domestic canids and preventative measures will become a priority., Graphical abstract Image 1, Highlights • We detected Em infections in wolves and shepherd dogs 130 km south of the southern-most case reported so far in the European Union. • Em infections in wolves and dogs in this area may indicate a range expansion and the establishment of a new endemic area. • This area is along an ecological corridor connecting the Alps to the Apennines, with the potential for further expansion. • It is priority to implement surveillance on the local fox population, and inform practitioners of the increased risk.

Summary Cystic echinococcosis (CE) and alveolar echinococcosis (AE) are considered as one of the most important zoonotic diseases in Romania, where they are subject to mandatory reporting. To obtain more knowledge about the genetic diversity of Echinococcus causative agents of these diseases, 11 isolates from humans and ungulate intermediate hosts from the two regions of Romania were genotyped using mitochondrial markers. In clinical samples of five patients from north-eastern Romania (Iasi, Botosani, Vaslui counties), Echinococcus multilocularis was identified as causal agent by cox1 sequence analysis. To the best of our knowledge this finding presents the first molecular evidence of E. multilocularis in humans from Romania. Only two cases of AE in patients were previously documented in the country by serological methods. In our four patients the most widespread European variant E5 of E. multilocularis was recorded, whereas in isolate from Vaslui county three nucleotide substitutions were detected as compared to the most related E5 haplotype. One of these mutations (411T/G) matched N1 and N2 haplotypes described previously from North America. In six CE samples retrieved from western Romania (Caras-Severin and Timis counties), two human isolates were diagnosed as Echinococcus canadensis G7, one as E. granulosus s.s. G1 and one as E. granulosus s.s. G3 using atp6 and rrnS sequencing. In ungulates, the cattle isolate was allocated to E. granulosus s.s. G1 and pig isolate to E. canadensis G7. The two G7 findings in humans reinforced the recent view that G7 was underestimated as compared to the E. granulosus s.s. regarding human CE threat that can be further employed for identifying sources of infections and establishing suitable preventive measures.

Larval forms of the bot-fly Gasterophilus are obligate parasites commonly found in the gastrointestinal tract of equids, causing intestinal myiasis. Five species are reported so far in Italy, mostly observed during necroscopy, located in different portion of gastrointestinal tract of equids: G. intestinalis, G. nasalis, G. inermis, G. pecorum and G. haemorrhoidalis. An unusual finding of larval Gasterophilus intestinalis deeply inserted into the diaphragmatic muscle is here reported. Due to the uncommon localization, to the absence of clinical signs related to myiasis and subsequent uncertainty of species identity, identification was performed using an integrative taxonomical approach combining morphology with molecular tools for confirmatory reasons. This finding adds information on migration patterns of erratic larval forms in G. intestinalis, a feature of interest as gasterophiliasis is among the less studied intestinal myiasis of horses.

Alveolar echinococcosis, caused by the larval stages of the tapeworm Echinococcus multilocularis (Leuckart, 1863), is of increasing concern in the northern hemisphere. Most cases of alveolar echinococcosis (excluding Alaska) appear to be linked with European and Asian genotypes that highlight the need for a more precise delimiting of their actual distribution and tracing historical episodes of their translocations and introductions into new areas. We have herein summarized previous available research studies, which mentioned firstly described geographic M1/M2 variants of E. multilocularis using molecular tools (established by sequencing of mitochondrial genes cox1, 366 bp and nad1, 471 bp), in an attempt to consolidate their correct affiliations with the geographic origin in sense of the original description from the early 1990´s. Since 2009, inverted designations (M1 named as M2 and vice versa) are being prevailing in research literature (we found ten erroneous vs. three correct classifications) that might bias genetic interpretation of comparative data in specific cases. When comparing M1/M2 profiles to those obtained from mitochondrial evidences over the last decades, the phylogenetic analysis revealed that the M1 strain (originally described from China, Alaska, North America) grouped with the Asian clade of E. multilocularis more recently established, whereas the M2 strain (described from the German vole) had a specific structure, in cox1 clustering with the North American clade. It is presumed that events of intercontinental expansion and isolation covering glacial and interglacial periods during the late Pleistocene have likely accounted for the transmission of this discrete genotype from Beringia into endemic area of western and central Europe via circumpolar movements of foxes.

Summary The occurrence of zoonotic parasitic nematodes in Atlantic chub mackerels (Scomber colias syn. Scomber japonicus) from Libyan waters was investigated, using epizootiological estimations and molecular specific characterization of larvae. Nematodes belonging to Anisakis spp., the main etiological agent of anisakiasis in Mediterranean waters, and to Hysterothylacium spp. so far considered not pathogenic to humans, were detected. Prevalence values were generally high in visceral cavities (over 40 % for both parasites) while were low for Anisakis (around 1 %) and null for Hysterothylacium in muscles. Moreover, the level of infections was associated with seasons, a feature potentially useful to plan fishing captures and to elaborate risk mitigation strategies for anisakiasis. Species molecular identification performed on a subsample described the presence of Hysterothylacium aduncum as the predominant species, along with Anisakis pegreffii and the hybrids (A. pegreffii and A. simplex sensu stricto), thus posing a concrete zoonotic risk following the consumption of such fish species as a raw preparation.

Nematodes belonging to the Trichuris genus are prevalent soil-transmitted helminths with a worldwide distribution in mammals, while humans are mainly affected in areas with insufficient sanitation such as in Africa, Asia and South America. Traditionally, whipworms infecting primates are referred to Trichuris trichiura, but recent molecular and morphological evidence suggests that more than one species may be able to infect humans and non-human primates. Here, we analyzed the genetic diversity and phylogeny of Trichuris infecting five different non-human primate species kept in captivity using sequencing of three mitochondrial genes (cox1, rrnL and cob). Phylogenetic analyses of both single and concatenated datasets suggested the presence of two main evolutionary lineages and several highly supported clades likely existing as separate taxa. The first lineage included Trichuris infecting the mantled guereza (Colobus guereza kikuyensis), the chacma baboon (Papio ursinus) and the green monkeys (Chlorocebus spp.), clustering together with Trichuris suis; the second lineage included Trichuris infecting the Japanese macaque (Macaca fuscata) and the hamadryas baboon (Papio hamadryas), clustering together with Trichuris spp. infecting humans. These results were supported by the genetic distance between samples, which suggested that at least two taxa are able to infect macaques, baboons and humans. The present study improves our understanding of the taxonomy and evolutionary relationships among Trichuris spp. infecting primates. It moreover suggests that multiple Trichuris spp. may circulate among host species and that Trichuris in non human primates (NHPs) may be zoonotic. Further studies are important to better understand the epidemiology of Trichuris in primates and for implementing appropriate control and/or conservation measures.

Members of the genera Anisakis and Pseudoterranova are the main causative agents of human anisakidosis: the disease is worldwide distributed, with major impact in countries with a large consumption of raw fish. Because of unspecific symptoms and limited diagnostic tools, incidence and burden of disease are probably underestimated. In Italy, all human infestations where the etiological agent has been properly identified, have been associated to the parasitic species Anisakis pegreffii, the most frequent anisakid in the Mediterranean area. Here, an invasive human case of pseudoterranoviasis is described for the first time in Italy: in 2015, a woman was found infected during a colonoscopy scheduled after the occurrence of nonspecific clinical symptoms. The nematode was found penetrating the ascending colon. The identification was performed by sequencing the mitochondrial region cox2 and by comparison to GenBank retrieved material using the BLAST search tool. The sample showed a 99% identity with Pseudoterranova decipiens sensu stricto. The record underlines the potential risk due to the consumption of raw or undercooked imported fishes.

The accurate identification of anisakid nematodes at any life cycle stage is important both to deepen the knowledge on their taxonomy, ecology, epidemiology and for diagnosis and control, as larval stages cause a clinical disease in humans known as anisakidosis. With the aim to investigate the presence of anisakid larvae, specimens of horse mackerel, Trachurus trachurus (Linnaeus, 1758), silver scabbardfish, Lepidopus caudatus (Euphrasen, 1788), European anchovy, Engraulis encrasicolus (Linnaeus, 1758) and opah fish, Lampris guttatus (Brunnich, 1788), were collected by trawling at depths ranging from 50 to 400 m. A molecular approach based on restriction profiles obtained after digestion of the nuclear ribosomal ITS region was used to identify Anisakis spp. larvae recovered in fish samples. Restriction profiles showed three banding patterns, corresponding to Anisakis pegreffii, Anisakis physeteris and to heterozygote pattern between A. pegreffii and Anisakis simplex s.s. Specimens showing the heterozygote restriction pattern were also analyzed by sequencing of the entire ITS region, to confirm the heterozygote status.

In the present study, we sequenced the complete mt genome (14,022 bp) of parasitic nematode Contracaecum rudolphii B and its structure and organization compared with Anisakis simplex s.l. The mt genome of C. rudolphii B is slightly longer than that of A. simplex s.l. (13,916 bp). C. rudolphii B mt genome is circular, and consists of 36 genes, including 12 genes for proteins, 2 genes for rRNA and 22 genes for tRNA. This genome contains a high A+T (70.5%) content. The mt gene order for C. rudolphii B is the same as those for A. simplex s.l., but it is distinctly different from other nematodes compared. The start codons inferred in the mt genome of C. rudolphii B are TTG and ATT. Six protein-coding genes use TAA as a stop codon whereas five genes use T and one genes use TAG as a termination codon. This pattern of codon usage reflects the strong bias for A and T in the mt genome of C. rudolphii B. Phylogenetic analyses using concatenated amino acid sequences of the 12 protein-coding genes, with three different computational algorithms (Bayes, ML and MP), all revealed distinct groups with high statistical support, indicating that C. rudolphii B and A. simplex s.l. is distinct but closely related species. These data provide additional novel mtDNA markers for studying the molecular epidemiology and population genetics of the C. rudolphii B, and should have implications for the molecular diagnosis, prevention and control of anisakidosis in humans and animals.

Larval forms of the genus Anisakis were reported infecting several fish species from the North African coasts of central Mediterranean Sea. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was used to investigate the occurrence of larval forms of different Anisakis species in teleost fishes and squid from North African coasts of the Mediterranean Sea and to establish the geographical and host range of these parasites in this area. A total of 282 Anisakis larvae were identified by PCR-RFLP from 13 teleost fish species and one cephalopod species captured at different sites off the Algerian, Tunisian and Libyan coasts. The type I larvae were found with a frequency of 93.62% and were identified as belonging to the following species: Anisakis simplex s.str., Anisakis pegreffii, A. simplex s.str/A. pegreffii hybrids and Anisakis typica. The type II larvae were found to belong to Anisakis physeteris, with the frequency of 6.38%. The record of A. simplex s.str/A. pegreffii hybrids, previously recorded from the Spanish and Portuguese Atlantic coasts and the Alboran Sea, extends their geographic distribution to the Tunisian coasts. The occurrence of A. simplex s.str. and hybrids away from their known area of distribution may predict the successful use of Anisakis larvae for tagging Scomber scombrus fish stocks for fisheries management purposes. Moreover, the results reported provide valuable information regarding the diversity of Anisakis species in the study area, indicating that several Anisakis sibling and morphospecies coexist in the North African coasts of the Mediterranean Sea.

Numerous studies have provided evidence that Echinococcus granulosus exists as a complex of different strains, that differ in a wide variety of criteria that have an impact on the epidemiology, pathology and control of cystic hydatid disease (CHD) and, to date, 10 distinct genotypes (G1-G10) have been identified. In Italy, sequence analysis of the mitochondrial cox1 and nad1 genes showed the occurrence of the G1 genotype, the common sheep strain, the G3 genotype, the buffalo strain and of one isolate identified as G2 genotype, the Tasmanian sheep strain. In the present work, we have analysed E. granulosus strains in Italy, by genotyping a large sample of isolates and by checking out the genetic differentiation within and among the G1 and G3 genotypes using an additional mitochondrial gene as marker, the rrnS gene. Sequencing of the rrnS gene revealed a significant genetic differentiation between isolates identified as belonging to the G1 and G3 genotypes, with fixed nucleotide substitutions. This study provides further evidence of the occurrence of the E. granulosus G3 buffalo strain in Italy, a strain previously thought to be confined to the Indian region.

The Parasitological Institute of the Slovak Academy of Sciences and the Section of Parasitology (formerly Institute of Parasitology) of the Department of Public Health Sciences of the Sapienza University of Rome have had a long term collaboration within several projects, starting with the first visiting fellowships in 1992 in Rome. Since then, the collaboration between the two institutions has continuously developed and has been focused mainly on the systematics of cestodes, starting in particular with the studies on the genetic polymorphism of fish Proteocephalus tapeworms in different host populations examined by enzyme electrophoresis. Some molecular studies have been later carried out on Eubothrium spp. and Echinococcus multilocularis, and most of our joint interest has been focused on Echinococcus granulosus and more recently, on Ascaris nematodes. The major achievements obtained during these years of collaboration are herein reported.

Background Although chamois Rupicapra spp. are the most abundant mountain ungulates in Europe, no data are available on the presence of Giardia duodenalis infecting these species. Methods A total of 157 fecal samples from Alpine Rupicapra rupicaprarupicapra and Apennine Rupicapra pyrenaicaornata chamois were tested for the presence of G. duodenalis by immunofluorescence test, quantitative Real Time PCR and end-point PCR for genotype characterization. Results G. duodenalis was detected in R. r. rupicapra and R. p. ornata, with a percentage value of 4.45 (5.82 and 1.85 %, respectively), and a cyst burden of up to 31,800 cysts/g of feces. Assemblages A/AI and E were identified in R. r. rupicapra and assemblage A/AIII in R. p. ornata. Conclusions The present study represents the first record of Giardia duodenalis in Rupicapra spp., suggesting that these wild bovids can play an epidemiological role in environmental contamination and transmission of both zoonotic and non-zoonotic genotypes.

After the publication of this work [1], we noticed an error in the Methods Section. The reported sequence of the ProSc “5′-GGTAACTTGTATGAAGGGACTAACTAAA-3′” is not the correct one. The correct one is “ProSc: 5′-GGTAACTTGTATGAAGGRAYTAACTAAA-3′”., This study was funded by i. the Italian Ministry of University and Research (MIUR), ii. the Project PRIN Genomics and host-pathogen interactions: a model study in the One-Health perspective, and iii. the Deanship of Scientific Research at King Saud University through the research group (project number RGP_020).

[Background] The mite Sarcoptes scabiei has a known host-range of over 100 mammal species including humans. One of the prime objectives of the Sarcoptes-World Molecular Network (WMN) is to design and develop universal Sarcoptes PCR-based diagnosis methods., [Methods] We describe here for the first time two universal mitochondrial-based diagnosis methods: (i) conventional end-point PCR and (ii) TaqMan real-time PCR. The design of both of these universal diagnosis methods was based on Sarcoptes samples collected from 23 host species in 14 countries., [Results] These methods, based on skin scrapings, were successfully used to etiologically confirm the diagnosis of different clinical degrees of sarcoptic mange in 48 animals belonging to six species. These universal PCR-based diagnosis methods are highly specific, technically sensitive and simple, and are based on the amplification of 135 bp from the Mitochondrial 16S rDNA. The method based on TaqMan real-time qPCR was more sensitive than the conventional end-point PCR., [Conclusions] Two universal PCR-based diagnosis methods for S. scabiei were successfully designed and applied; one based on conventional end-point PCR and the other on TaqMan real-time PCR. We recommend further testing and the application of these new universal methods worldwide., This study was funded by i. the Italian Ministry of University and Research (MIUR), ii. the Project PRIN Genomics and host-pathogen interactions: a model study in the One-Health perspective, and iii. the Deanship of Scientific Research at King Saud University through the research group (project number RGP_020).

Nucleotide sequencing of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene and isoenzyme analysis were used to survey the genetic variability in Echinococcus multilocularis populations from Slovakia. A sample of 12 isolates acquired from 10 different districts from red foxes exhibited identical sequences. Compared with the previously described E. multilocularis variants, one base substitution was consistently observed relative to the M1 variant (detected in China, Alaska, North America, Japan) and three base substitutions were recorded relative to the M2 variant (detected in Germany) in the CO1 fragment. These data, along with the recently gathered data from French isolates, are indicative of a genetically unique population occurring in Central and Western Europe. Electrophoretic examination of enzymes produced by 14 gene loci revealed intraspecific polymorphism only with the glucose-phosphate isomerase (two distinct patterns) and the mannosephosphate isomerase (four genotypes composed of three alleles) enzyme systems. To allow a fast species differentiation of E. multilocularis and E. granulosus (specifically, the G7 genotype occurring in Slovakia), discriminative electrophoretic characters between the species were obtained by isoenzyme analysis. Fixed genetic differences between the species were detected in the glucose-phosphate isomerase, esterase and aldolase systems, and partial differences were detected in four additional systems.

Larval stages of Anisakis spp. (Nematoda: Anisakidae) were found encapsulated or free in the viscera and abdominal cavity of the black-scabbard fish, Aphanopus carbo, chub mackerel, Scomber japonicus and oceanic horse mackerel, Trachurus picturatus in Madeiran waters. The prevalence of infection reached 97.2% (n=142) for A. carbo, 69.5% (n=154) for S. japonicus and 62.5% (n=40) for T. picturatus. Considerable differences in parasite intensities between A. carbo and both S. japonicus and T. picturatus were found, with mean intensities up to 69.6 in A. carbo, while in the other two fish hosts the intensity reached only a maximum of 2.6. These differences were probably due to different feeding behaviours of the hosts. Intensities of Anisakis sp. in A. carbo were high irrespective of sex and season. No relationship between host length and prevalence of infection was observed for A. carbo, while for S. japonicus a weak positive significant relationship was found.

Investigations of microbiota within mosquitoes continue to widen the spectrum of possible symbiont-based applications against vector-borne diseases. In this context, α-proteobacteria of the genus Asaia (Rhodospirillales: Acetobacteraceae) are emerging as possible endosymbiotic candidates, particularly in paratransgenic approaches aimed at interrupting pathogen transmission. Previous studies have shown that Asaia spp. distribution among Anopheles gambiae and Anopheles stephensi (Diptera: Culicidae) mosquitoes displayed positive rates of infection in isolated midguts, salivary glands and reproductive tissues. Similarly, Asaia has been detected in Aedes albopictus (Stegomyia albopicta) and Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae) populations. Within the Culex pipiens complex (Diptera: Culicidae), Asaia infection is still largely unexplored. Here, we summarize a preliminary survey of laboratory-reared Cx. pipiens complex and field-collected Culex quinquefasciatus for the presence of Asaia spp., and present the first identification of Asaia in some of the members of the Cx. pipiens complex and the first description in West African populations of Cx. quinquefasciatus.

The genus Anisakis includes nine recognized species and the complex of cryptic species Anisakis simplex s. l. is often associated with the human disease known as anisakiasis. During the last decades the use of nuclear ribosomal ITS allowed the identification and description of numerous anisakid nematodes and the discovery of recombinant genotypes or putative hybrids even in other parasitic helminths, such as those between A. simplex sensu stricto and A. pegreffii. The existence of pure hybrids of the two sibling species has been long debated due to the large recovery of larval forms from sympatric areas and the rare observation of adult hybrids. The aims of the present report were to identify anisakid nematodes collected from Stenella coeruleoalba using PCR-RFLP of ITS and to focus the interest on hybrid forms using a High Resolution Melting (HRM) and direct sequencing analyses, since the new record of putative hybrid at adult stage. The PCR-RFLP analysis enabled to identify A. simplex s.s., A. pegreffii, the heterozygous genotype of the two species and A. physeteris. The use of the genotyping approach based on HRM confirmed the profiles of the two species A. simplex s.s. and A. pegreffii, and of the hybrid individuals. The new record of adult hybrids in definitive hosts rekindles the long debate about their existence and their evolutionary meaning. Since the reproductive isolation between A. simplex s.s. and A. pegreffii is the assumption for their existence as separated species, the use of alternative molecular markers and population genetic studies on adult anisakids are recommended.

The second internal transcribed spacer (ITS2) of the ribosomal RNA gene array of 6 species of diplozoids; Eudiplozoon nipponicum, Paradiplozoon bliccae, P. homoion, P. megan, P. pavlovskii and P. sapae, was amplified by PCR and sequenced. These sequences clearly demonstrate discrimination at the species level and confirm the validity of species determined by morphological identification. No intraspecific variation was found in the ITS2 sequences. There were no differences in the ITS2 sequences of P. homoion from populations parasitizing different host species. The length of the PCR product allowed discrimination of E. nipponicum from the Paradiplozoon species. Digestion of the amplified ITS2 fragment with enzymes AluI, HaeIII and HinfI provided useful genetic markers for species identification. The genetic relationships between diplozoids again demonstrated that E. nipponicum was the most genetically distinct species, whereas P. bliccae and P. sapae were the species most closely related. This represents the first molecular taxonomic study of these interesting parasites and demonstrates the utility of these methods for addressing questions of systematics.

Nuclear-encoded large-subunit ribosomal DNA sequences were used to infer a phylogenetic hypothesis for 17 taxa (16 nominal species) of the genera Contracaecum and Phocascaris. Phylogenetic trees based on these data have been used to assess the validity of the taxonomic distinction between these genera, which was based on the presence or absence of certain structural features, rather than on explicit hypotheses of evolutionary history. Phylogenetic hypotheses based on parsimony, likelihood, and neighbor-joining analyses of these sequence data strongly support the hypothesis that species of Phocascaris are nested within the clade of Contracaecum species hosted by phocid seals, and are more closely related to species of the Contracaecum osculatum complex than to other Contracaecum species. Alternative tree topologies representing Phocascaris as not nested within the C. osculatum complex were significantly worse interpretations of these sequence data. Phylogenetic analysis also provides strong support for the monophyly of all taxa (Contracaecum and Phocascaris) from phocid seals, which is consistent with Berland's (1964) proposal that such species form a natural group; however, his proposal to recognize all species in phocid seals as Phocascaris, with all species from birds as Contracaecum would result in a paraphyletic Contracaecum, according to the molecular phylogenetic hypothesis.

Variability in Echinococcus granulosus is very important epidemiologically since strain characteristics may influence local patterns of transmission of hydatid disease. To classify the genotype presented in pig protoscoleces of the Slovak territory, a DNA-based approach has been used. Nucleotide sequences for a 471 bp region of the mitochondrial NADH dehydrogenase 1 (ND1) gene revealed a substantial affinity of isolates examined to the G7 genotype. Only a 0.9–3.4% sequence variation was recorded for E. granulosus samples compared with the reference G7 variant. To distinguish between G7 and G9 genotypes not differing in ND1 sequences, isolates were additionally examined by PCR-RFLP analysis of the nuclear ITS1 region. The resulting two-banded pattern is characteristic for the G7 strain. The data presented thus provides the first explicit evidence of the G7 genotype in the Slovak region.

Polymerase-chain-reaction-based restriction fragment length polymorphism analysis was performed to establish genetic markers in rDNA, for the identification of the three sibling species of the Anisakis simplex complex and morphologically differentiated Anisakis species, i.e. Anisakis physeteris, Anisakis schupakovi, Anisakis typica and Anisakis ziphidarum. Different restriction patterns were found between A. simplex sensu stricto and Anisakis pegreffii with two of the restriction endonucleases used (HinfI and TaqI), between A. simplex sensu stricto and A. simplex C with one endonuclease (HhaI), and between A. simplex C and Aniskis pegreffii with three endonucleases (HhaI, HinfI and TaqI), while no variation in patterns was detected among individuals within each species. The species A. physeteris, A. schupakovi, A. typica and A. ziphidarum were found to be different from each other and different from the three sibling species of the A. simplex complex by distinct fragments using 10-12 of the endonucleases tested. The polymorphisms obtained by restriction fragment length polymorphisms have provided a new set of genetic markers for the accurate identification of sibling species and morphospecies.

Enzyme electrophoresis has been used to examine genetic diversity in a population ofProteocephalus exiguusLa Rue, 1911 (Cestoda: Proteocephalidae), parasitizing salmonid and coregonid fishes. Among 16 loci tested, three polymorphic loci (Ada, Got, Pgm-2) were found. Six different genotypes at theGotlocus distributed in Hardy-Weinberg equilibrium suggest remarkable genetic flexibility ofP. exiguus. Balancing selection is proposed as the mechanism maintaining genetic variation within the species. Data of genetic variability parameters (Ho= 0.064; He= 0.07; P = 0.19) and outcrossing rate (t = 0.842) ofP. exiguuspopulation have been provided. Possible factors affecting these data are discussed.

DNA sequencing of the nuclear ribosomal DNA internal transcribed spacers (ITS) and mitochondrial rrnS and cox2 genes, and analysis of polymorphisms in restriction profiles in the ITS and rrnS, were used to characterise anisakid nematodes belonging to Contracaecum Railliet & Henry, 1912 infecting the brown pelican Pelecanus occidentalis (L.) in Galveston Bay, Texas and Sarasota Bay, Florida. Molecular data led to the detection of two new species: Contracaecum fagerholmi n. sp., which was also supported by clear morphological evidence, and Contracaecum rudolphii F, a new cryptic species within the Contracaecum rudolphii Hartwich, 1964 complex. Bayesian phylogenetic analysis demonstrated that C. fagerholmi and C. rudolphii F form two well-separated clusters, with C. fagerholmi being closely related to Contracaecum bioccai Mattiucci et al., 2008 and C. rudolphii F being included in the C. rudolphii complex. C. fagerholmi can be readily differentiated morphologically from all of its congeners, other than C. microcephalum (Rudolphii 1809) and the five currently recognised members of the C. rudolphii complex (C. rudolphii A, B, C, D and E). C. fagerholmi differs from C. microcephalum in the length of the spicules and the shape of the distal tip of the spicules, and from C. rudolphii (sensu lato) in the shape and size of the ventro-lateral and dorsal lips and by having interlabia which are not distally bifurcate. Further studies are needed to determine which morphological characteristics can be used to distinguish the cryptic species of the C. rudolphii complex in order to assign them with formal names. The recovery of a third species, C. bioccai, from the brown pelican confirms its occurrence in this host and extends its known geographical distribution.

In the present study, 407 anisakid nematodes, collected from 11 different species of cetaceans of the families Delphinidae, Kogiidae, Physeteridae, and Ziphiidae, from the southeastern Atlantic coasts of USA, the Gulf of Mexico, and the Caribbean Sea, were examined morphologically and genetically characterized by PCR restriction fragment length polymorphism to identify them to species level, assess their relative frequencies in definitive hosts, and determine any host preference. Sequence data from nuclear ribosomal internal transcribed spacer and mitochondrial cox2 genes were analysed by maximum parsimony and Bayesian inference methods, as separate and combined datasets, to evaluate phylogenetic relationships among taxa. The results revealed a highly diverse ascaridoid community. Seven Anisakis species and Pseudoterranova species were recovered as adult parasites. Larval forms of Contracaecum multipapillatum were also found in a coastal population of bottlenose dolphins. The phylogenetic trees obtained from the combined dataset (and most individual datasets) revealed the existence of distinct clades, the first including species of the Anisakis simplex complex (A. simplex s.s., Anisakis pegreffii, A. simplex C), (Anisakis nascettii, Anisakis ziphidarum) and the second including Pseudoterranova ceticola ((Anisakis paggiae, (Anisakis physeteris, Anisakis brevispiculata)). This finding, excluding the relationship of P. ceticola, is consistent with the morphology of adult and larval specimens. Considering the presence versus absence of an intestinal cecum, the relationship of P. ceticola with the members of the second clade of Anisakis appears inconsistent with morphological evidences but consistent with host preference. The position of Anisakis typica as the sister group to the two main anisakid clades indicates that it represents a third distinct lineage.

Eleven parasite taxa were found infecting 68 Atlantic chub mackerel, Scomber colias Gmelin, 1789 from the Canary Islands, Central North Atlantic. The most abundant parasites were the gill monogenean Pseudokuhnia minor (P = 54.4%), larval anisakid nematodes (P = 11.8%) in the body cavity, a larval tetraphyllidean infecting bile ducts (P = 8.8%) and didymozoid digeneans infecting the gills (P = 7.4%). No correlation between fish length and abundance of infection with these parasites was found. Within the Atlantic, the comparison of present results with previous reports on the occurrence of parasites in this fish host, might suggest that there is more than one population unit of Atlantic chub mackerel in the Eastern Atlantic., Sí

Genetic variation within and between population samples from 22 locations of the Atlantic Arctic-Boreal region, including 1657 specimens morphologically assigned to Contracaecum osculatum, was electrophoretically analysed at 17 loci. Highly significant deviations from the Hardy-Weinberg equilibrium were found at various loci in several samples, owing to the existence of three distinct gene pools within C. osculatum (sensu lato) from the study area. These gene pools correspond to three biological species (provisionally designated A, B and C), characterized by distinct genotypes at several diagnostic loci. Reproductive isolation between C.osculatum A, B and C is confirmed by the lack of F1, recombinant, or backcross genotypes in sympatric areas, despite the occurrence of multiple infections. Mean heterozygosity per locus is on average 0.11 in species A, 0.10 in B and 0.07 in C. High levels of gene flow were found within each of the three species, the values of Nm (number of migrant individuals) ranging from 3.41 (C. osculatum C) to 5.77 (C. osculatum A). Average Nei's genetic distance is 0.46 between A and B, 0.50 between A and C and 0.77 between B and C. From these values, times of evolutionary divergence from 2 to 4 million years can be estimated. Genetic relationships among populations and species of the C. osculatum complex are illustrated by principal component analysis. The role of both geographical isolation and host preferences in the speciation of C. osculatum (sensu lato) is discussed. A morphological distinction of the three species has not yet been possible (sibling species). However, there is evidence that the name C. osculatum (sensu stricto) should be used for species C, which shows a geographical distribution and definitive host corresponding to the neotype of C. osculatum (sensu stricto). Finally, a comparison is made between the members of the C. osculatum complex from the Atlantic Arctic-Boreal region and those of the Pseudoterranova decipiens complex from the same area, as to: (i) times of evolutionary divergence, (ii) geographical distribution, and (iii) host preferences.

Genetic variability of the ovine parasiteHaemonchus contortusfrom the Alpine area was investigated using mitochondrial DNA (nd4gene), internal transcribed spacers 1 and 2 and microsatellites, in order to assess whether cross-transmission between domestic and wild ruminants occurs. The dataset was composed of 78 individual adult maleH. contortuscollected from chamois (Rupicapra r. rupicapra), roe deer (Capreolus capreolus), alpine ibex (Capra ibex ibex), domestic goat (Capra hircus) and sheep (Ovis aries) from different alpine areas. The data obtained show low host specificity and high genetic variation withinH. contortuspopulations. The analyses indicate the presence of two mitochondrial haplotype clusters among host species and the absence of cryptic parasite species, confirmingH. contortusas a generalist nematode and suggesting that parasite transmission between populations of domestic and wild ruminants normally occurs.

Twenty-three adults (only one male) and two fourth-stage larvae of Anisakis, recovered from the stomach of a Blainville's beaked whale (Mesoplodon densirostris) stranded in Galicia (NW Spain), were studied morphologically and molecularly. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing studies carried out on the ribosomal DNA spacers and on the cox2 mitochondrial gene confirm that these nematodes belong to the taxon Anisakis sp. A, which was previously detected as a larval stage in fishes from Madeiran waters. In addition, our molecular studies demonstrate that Anisakis sp. A and the taxon Anisakis sp., previously found in other Mesoplodon spp., are the same species. The adults of Anisakis sp. A are morphologically similar to A. ziphidarum but with a shorter body length and longer spicules (right spicule 2.42 mm, left spicule 2.30 mm). Since the poor condition of the male tail cuticle prevented a proper description of the caudal plates and the pattern of caudal papillae, we propose to retain the name Anisakis sp. A until new males are described correctly.

A total of 242 larval forms of Anisakis collected from marine fishes at different sites off the Moroccan and Mauritanian coasts, recognised as belonging to Type I and Type II larvae, were identified by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphisms) of the ITS (Internal Transcribed Spacers) region (ITS-1, 5.8 subunit rRNA gene and ITS-2), using a previously established molecular key. The Type I larvae were found with a frequency of 98.34% and were identified as belonging to the following species: A. simplex s.str., A. pegreffii, A. simplex s.str/A. pegreffii heterozygote genotypes, A. typica, A. ziphidarum and Anisakis sp. A. The Type II larvae were found to belong to A. physeteris, with the frequency of 1.65%. The results reported in the present study provide further epizootiological and biological data on the Anisakis spp. in marine fishes off the Moroccan and Mauritanian coasts, improving the picture of the occurrence of these species in the central Atlantic coasts.

Une chatte de 10 ans a ete apportee a l'Istituto Zooprofilattico Sperimentale delle Regioni iazio e Toscana pour un examen post-mortem. l'animal examine vivait au milieu d'une colonie de 26 chats, sur la terrasse d'un appartement a Rome, au huitieme etage, et etait nourri avec des aliments en boite pour chat l'anamnese a mis en evidence des troubles de l'equilibre, des vomissements et convulsions, ceci pendant deux jours, le tout suivi d'une mort subite. A la necropsie, la chatte presentait les signes suivants: rhinite mucoide, tracheite sanieuse, zones pneumoniques circonscrites, taches foncees au niveau du foie, gastrite catarrhale hemorragique, enterite fibreuse et hyperhemie meningee. Les cavites thoraciques et abdominales etaient entierement envahies par des centaines de cestodes au stade larvaire. On a pu constater la presence des memes parasites dans des nodules des parenchymes pancreatique, pulmonaire et renal. L'analyse au microscope a permis d'identifier des stades larvaires (metacestodes) de cestodes du genre Mesocestoides. l'analyse moleculaire des metacestodes indique un rapport etroit avec des membres du genre Mesocestoides, bien qu'une variation significative ait ete trouvee en ce qui concerne les donnees disponibles d'autres especes du genre.

Anisakid nematodes belonging to the Anisakis simplex complex are highly prevalent in several fish species off the coast of Portugal and are an important zoonotic problem in the Iberian Peninsula. Two reproductively isolated sibling species of the Anisakis simplex complex were identified from Pleuronectiformes inhabiting the Portuguese coast using restriction fragment length polymorphism (RFLP). Recombinant genotypes corresponding to presumptive Anisakis simplex sensu stricto and Anisakis pegreffii hybrids were also detected by this technique, as well as the species Anisakis typica. Although 25 species of Pleuronectiformes were investigated, Anisakis spp. larvae were only found in seven: Arnoglossus imperialis, Arnoglossus laterna, Lepidorhombus boscii, Citharus linguatula, Platichthys flesus, Dicologlossa cuneata and Solea senegalensis. The occurrence of hybrids in relatively sedentary fishes such as the Pleuronectiformes suggests that the Portuguese coast may constitute an area of hybridization and, therefore, is of particular interest for the study of the process of hybridization and speciation for these anisakids.

One-hundred and fifteen anisakid larvae from 3 different fish hosts, Aphanopus carbo, Scomber japonicus, and Trachurus picturatus, caught in Madeiran waters, were identified by PCR-RFLP. Three distinct species were identified in A. carbo, namely Anisakis simplex sensu srricto, Anisakis pegreffii, and Anisakis ziphidarum; 5 in S. japonicus, i.e., A. simplex s.s., A. pegreffii, Anisakis physeteris, Anisakis typica, and A. ziphidarum; and 3 in T. picturatus, i.e., A. simplex s.s., A. pegreffii, and A. typica. Anisakis simplex s.s. was the most frequent species in both A. carbo and S. japonicus (54% and 23.5%, respectively). Anisakis pegreffii and A. physeteris occurred with a frequency of 20.6% in S. japonicus, whereas in T. picturatus the most frequent species was A. typica (41.9%), followed by A. simplex s.s. (32.3%). Furthermore, A. carbo and S. japonicus were infected by an apparently undescribed taxon, provisionally named Anisakis sp. A. Based on estimations of the genetic distance, this new taxon seems to be more similar to A. ziphidarum (0.0335) than to other species of the genus.

Individual specimens of Anisakis, Pseudoterranova, and Contracaecum collected from marine mammals inhabiting northern Pacific waters were used for comparative diagnostic and molecular phylogenetic analyses. Forty-eight new sequences were obtained for this study of 14 Anisakis taxa, 8 Pseudoterranova taxa, 4 Contracaecum taxa, and 4 outgroup species. Partial 28S (LSU) and complete internal transcribed spacer (ITS-1, 5.8S, ITS-2) ribosomal DNA was amplified by the polymerase chain reaction and sequenced. Sequences of ITS indicated that Pseudoterranova specimens from Zalophus californianus (California sea lion), Mirounga angustirostris (northern elephant seal), Phoca vitulina (harbor seal), Enhydra lutris (sea otter), and Eumetopias jubatus (Steller's sea lion) exactly matched P. decipiens s. str., extending the host and geographic range of this species. Anisakis from northern Pacific marine mammals were most closely related to members of the A. simplex species complex. Comparison of Anisakis ITS sequences diagnosed the presence of A. simplex C in 2 M. angustirostris hosts, which is a new host record. Anisakis specimens from Phocoena phocoena (harbor porpoise), Lissodelphis borealis (Pacific rightwhale porpoise), and E. jubatus included 3 ITS sequences that did not match any known species. Contracaecum adults obtained from Z. californianus were most closely related to C. ogmorhini s.l. and C. rudolphii, but ITS sequences of these Contracaecum specimens did not match C. ogmorhini s. str. or C. margolisi. These novel Anisakis and Contracaecum ITS sequences may represent previously uncharacterized species. Phylogenetic analysis of LSU sequences revealed strong support for the monophyly of Anisakinae, Contracaecum plus Phocascaris, Pseudoterranova, and Anisakis. Phylogenetic trees inferred from ITS sequences yielded robustly supported relationships for Pseudoterranova and Anisakis species that are primarily consistent with previously published phenograms based on multilocus electrophoretic data.

Specimens of Contracaecum rudolphii sensu lato (s.l.) (Nematoda: Anisakidae) from Phalacrocorax carbo sinensis from northeastern and central Italy were characterised genetically and compared with those from Phalacrocorax aristotelis from Galician coasts, Spain (identified as C. rudolphii A by multilocus enzyme electrophoresis) and with specimens of C. septentrionale from Alca torda from the Galician coasts, Spain. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified by polymerase chain reaction (PCR) from individual nematodes and the amplicons subjected to single-strand conformation polymorphism (SSCP) analysis and/or sequencing. For each ITS region, C. septentrionale specimens were distinct from those of C. rudolphii (s.l.) and C. rudolphii A based on SSCP profiles and ITS sequences. Some specimens of C. rudolphii (s.l.) had the same SSCP profiles and ITS sequences as C. rudolphii A, whereas the others had distinct SSCP profiles and ITS sequences and were suggested to represent C. rudolphii B based on host and geographical origins and genetic similarity to C. rudolphii A. While no length or nucleotide variation in the ITS-1 and ITS-2 sequences was detected within each taxon, nucleotide differences of 1.8-5.5% (ITS-1) and 5.1-12.2% (ITS-2) were detected among them. The results support the hypothesis that C. rudolphii represents a complex of at least two sibling species and provide support for the validity of C. septentrionale as a separate species. The definition of genetic markers in the ITS rDNA provides opportunities for investigating the life cycles, transmission patterns and ecology of the anisakid nematodes studied herein.

Monogeneans belonging to the genus Ligophorus Euzet & Suriano, 1977 are parasitic on the gills of several fish species of the family Mugilidae (mullets). At present, 12 species belonging to this genus have been described in the Mediterranean Sea and each host species is infected by a number of Ligophorus species that are not found on other mullets. A multivariate study using principal component and canonical variate analyses was carried out, with the aim of investigating the extent of morphometric variability within and between the 12 Mediterranean species of Ligophorus. Significant intraspecific morphometric variation was detected between two allopatric populations of L. angustus Euzet & Suriano, 1977 parasitic on Chelon labrosus from Sardinia; however, the existence of a new species of Ligophorus needs further evidence. Analysis of interspecific variation confirms the validity of the morphological keys used at present for the identification of Ligophorus at the species level and provides a picture of the relationships between species, indicating that the two species parasitising Mugil cephalus, L. chabaudi Euzet & Suriano, 1977 and L. mugilinus (Hargis, 1955) are the most differentiated. The hypothesis that strict host-parasite ecological relationships may be the result of co-speciation processes is briefly discussed.

The present study was undertaken to characterize more thoroughly aetiological agent of cystic echinococcosis that still represents a health problem in the Slovak Republic. Sequencing of the mitochondrial ND1 gene, Random Amplified Polymorphic DNA (RAPD) analysis and isoenzyme analyses were used to characterize genetically protoscolices of 37 isolates from pigs and cattle and two isolates from humans collected in different regions of the Slovak Republic. Solely the G7 genotype (pig strain) was identified by ND1 sequences in all 14 pigs isolate examined by this method. This genotype was also found in two human patients and in two cattle. The exclusive finding of the G7 variant has implications for the implementation of control strategies given its unique developmental and biological properties. Despite striking morphological variability, a low degree of the intra-strain variability was found in the Slovak Echinococcus granulosus isolates with the genetic methods used. This was particularly associated with the shape and size of hooks, number of testes and shape of ovaries and uterus. GPI and MDH enzymes were found suitable for the discrimination of G7 and G1 (=cosmopolitan sheep strain) variant.

Bacteria of the genus Wolbachia constitute a group of intracellular and maternally inherited micro-organisms that are widespread in arthropods, inducing several reproductive disorders such as cytoplasmic incompatibility in their hosts. Considering relevant biological implications related to the presence of Wolbachia in several insect orders, for example its potential role as mechanism for rapid speciation and as vehicle to drive genetic markers in wild populations of vectors of medical and veterinary interest, we carried out an extensive polymerase chain reaction survey to detect Wolbachia in several species of mosquito belonging to genera involved in the transmission of pathogens. Five species out of 26 tested have shown to be infected; for four of them this is the first evidence of the Wolbachia infection. A phylogenetic analysis was also performed, positioning the five Wolbachia strains in the phyletic subdivision B.

In order to investigate the extent of the genetic variation in the DNA sequences of Sarcoptes scabiei, mite populations collected on Alpine chamois ( Rupicapra rupicapra), Pyrenean chamois ( Rupicapra pyrenaica) and red fox ( Vulpes vulpes) from different localities of Italy and Spain were studied. Sequence analyses were carried out on the second internal transcribed spacer of the nuclear ribosomal DNA and on the 16S mitochondrial rRNA gene. ITS-2 sequences showed a higher degree of genetic polymorphism, mostly randomly distributed in the isolates from different hosts and localities, although both genomic regions are characterised by fixed nucleotide substitutions which were able to discriminate the mites collected on Pyrenean chamois from north-western Spain and on foxes from north-eastern Spain and from north-western Italy with respect to the other isolates. These results suggest the existence of a limitation to free gene exchange between the studied populations, probably related to the genetic structuring of local populations rather than to a differential adaptation to host species.

The anisakid nematodes of seals from different geographical origins, previously identified as Contracaecum osculatum A, C. osculatum B, C. osculatum C, C. osculatum D, C. osculatulm E and C. osculatum baicalensis by multilocus enzyme electrophoresis, were characterised using a DNA approach. The first and second internal transcribed spacers (ITS-1, ITS-2) of ribosomal DNA (rDNA) were individually amplified by polymerase chain reaction (PCR) and analysed by single-strand conformation polymorphism (SSCP) and sequencing. SSCP analyses allowed the unequivocal differentiation of all taxa except C. osculatum D from C. osculatum E. While C. osculatum D and C. osculatum E had identical ITS sequences, each of the other four taxa had distinct sequences, with interspecific differences ranging from 0.3% to 2.3%. C. osculatum C was genetically the most distinct taxon with respect to all other members of the species complex.

Isozyme analysis at 24 loci was carried out on anisakid nematodes of the Anisakis simplex complex, recovered from various intermediate/paratenic (squid, fish) and definitive (marine mammals) hosts from various parts of the world. A number of samples were found to belong to A. simplex sensu stricto and Anisakis pegreffii, widely extending the geographic ranges and the number of hosts of these 2 species. In addition, a new distinct gene pool was detected, showing different alleles with respect to A. simplex s. str and A. pegreffii at 5 diagnostic loci (99% level). Samples with this gene pool were assigned to a new species, provisionally labeled A. simplex C. Reproductive isolation between A. simplex C and the other 2 Anisakis species was directly assessed by the lack of hybrid and recombinant genotypes in mixed samples from sympatric areas, i.e., Pacific Canada for A. simplex C+A. simplex s. str., South Africa and New Zealand for A. simplex C+A. pegreffii, even when such samples were recovered from the same individual host. Similar levels of genetic divergence were observed among the three species (DNei from 0.36 to 0.45). At the intraspecific level, Canadian Pacific and Austral populations of A. simplex C were found to be genetically rather differentiated from one another (average DNei = 0.08), contrasting with the remarkable genetic homogeneity detected within both A. simplex s. str. and A. pegreffii (average DNei about 0.01). Accordingly, a lower amount of gene flow was estimated within A. simplex C (Nm = 1.6) than within the other 2 species (Nm = 5.4 and 17.7, respectively). Anisakis simplex C showed the highest average values of genetic variability with respect to both A. simplex s. str. and A. pegreffii, e.g., expected mean heterozygosity. Hr = 0.23, 0.16, and 0.11, respectively, in the 3 species. Data on geographic distribution and hosts of the 3 members so far detected in the A. simplex complex are given. Their ecological niche is markedly differentiated, with a low proportion of hosts shared. Intermediate and definitive hosts of A. simplex s. str. and A. pegreffii appear to belong to distinct food webs, benthodemersal, and pelagic, respectively; this would lead to different transmission pathways for the parasites.

Genetic variation of the Antarctic anisakid Contracaecum radiatum from the Ross and Weddell Seas is studied at 24 enzyme loci. All polymorphic loci proved to be in Hardy-Weinberg equilibrium, with no significant differences between the samples tested. Several loci were found to be diagnostic between C. radiatum and the five known members of the C. osculatum complex, indicating that no gene exchange occurs between them and confirming their specific status. No F1 hybrids, recombinant or introgressed individuals, sharing radiatum and osculatum alleles, were detected in the Antarctic Contracaecum samples; this directly proves the reproductive isolation of C. radiatum. Biochemical keys are given for the identification of Antarctic C. radiatum, C. osculatum D and E, both for adults (males and females) and larvae. Higher values of genetic variability were observed in these Antarctic Contracaecum species than in the Arctic-Boreal C. osculatum members, possibly related to a lower degree of habitat disturbance (i.e. by pollution, fishing and hunting) in the Antarctic region. On the basis of genetic distances, the evolutionary divergence between C. radiatum and C. osculatum (sensu lato) started about 5 million years ago, possibly with the first colonisation of the Antarctic region by seals. Data on paratenic (fish) and definitive (seal) hosts of C. radiatum and C. osculatum D and E are given.

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.

Biochemical keys based on the electrophoretic patterns of nine enzymatic loci diagnostic between Dirofilaria repens and D. immitis were used to examine an immature female of Dirofilaria removed from a 32-year-old woman resident in Caserta, Italy. The worm, tentatively assigned on a morphological basis to D. conjunctivae-D. repens, showed at all the loci examined the same electrophoretic pattern as an Italian dog strain of D. repens. This genetic evidence is in substantial agreement with the hypothesis, already supported by morphological and epidemiological data, that D. conjunctivae (Addario, 1885) Desportes, 1939-1940 and D. repens Railliet and Henry, 1911 should be considered as synonyms. According to the law of priority D. conjunctivae should be the valid name for the species; however, it is proposed to preserve the name repens, well-established for the dog subcutaneous filaria. Multilocus electrophoretic analysis appears to be a valuable tool for the identification of aetiological agents of human zoonotic filariae, particularly in areas where more than one species of the subgenus Nochtiella are present.

The distribution of two acanthocephalan species (Pomphorhynchus laevis and Acanthocephalus anguillae) in the chub (Leuciscus cephalus) was studied in foriver reaches characterized by different levels of pollution: the River Ticino ne Abbiategrasso (unpolluted), the Naviglio Grande Canal, in Milano (slightly polluted), the River Lambro near Merone village (polluted) and the Riv Lambro near Monza (severely polluted).Pomphorhynchus laevis was restricted to the unpolluted and the slight polluted sites, while the intensity of A. anguillae increased proportionally water pollution. These differences were partially explained by the variation in abundance of their intermediate hosts (Echinogammarus stammeri for P. lavis and Asellus aquaticus for A. anguillae). Data on the occurrence of P. laevis at A. anguillae showed a significant negative binomial frequency distributic suggesting their tendency to be aggregated within the host populations L. cephalus.