New cytogenetic data on Caryophyllaeus laticeps and Paracaryophyllaeus gotoi, parasites of evolutionary interest.

Caryophyllideans are intestinal parasites of freshwater fishes, occupying a basal position among the 'true' tapeworms. We performed detailed cytogenetic analyses of the well-known caryophyllidean species Caryophyllaeus laticeps. For comparison, we also examined for the first time the chromosomes of Paracaryophyllaeus gotoi, a specific parasite of loaches in China. Both species showed a diploid chromosome number of 2n = 20, n = 10m. Chromomycin A3 (CMA3)/diamidino-2-phenylindole (DAPI) staining performed for the first time in the class Cestoda revealed CMA3+/DAPI− bands in the pericentromeric regions of the short arms of chromosome pair no. 7 in the karyotype of C. laticeps. Fluorescence in situ hybridization with the 18S rDNA probe confirmed the presence of a single cluster of major rDNA near the centromere on a pair of small chromosomes in both species. These findings support the hypothesis that the ancestral state in the family Caryophyllaeidae is a single interstitial cluster of major rDNA genes and thus one nucleolar organizer region per haploid genome. Our results, which we presented together with literature data plotted on a phylogenetic tree, show stability of caryophyllidean karyotypes at the genus level, but showed differences between genera without a clear phylogenetic signal. The data allowed us to at least formulate a hypothesis about the ancestral haploid chromosome number of n = 10 for the family Caryophyllaeidae and possibly for the sister family Capingentidae. In addition, we compared two populations of C. laticeps from water bodies with different levels of polychlorinated biphenyl contamination, showing a slightly increased incidence of chromosomal abnormalities at the contaminated site.

Molecular cytogenetic analysis of a triploid population of the human broad tapeworm, Dibothriocephalus latus (Diphyllobothriidea).

The large-sized tapeworm Dibothriocephalus latus is known as the broad or fish-borne cestode of mammals that is capable to infect humans and cause diphyllobothriosis. Recently, molecular data on D. latus has been accumulating in the literature and a complete genome sequence has been published; however, little is known about the karyotype and chromosome architecture. In this study, an in-depth karyological analysis of 2 D. latus specimens was carried out. The plerocercoids originated from a perch caught in subalpine Lake Iseo (Italy) and the tapeworms were reared in hamsters. Both specimens contained cells with a highly variable number of chromosomes ranging from18 to 27. Nevertheless, the largest portion of mitotic figures (47%) showed a number corresponding to the triploid set, 3n = 27. Accordingly, the karyotype of the analyzed specimens consisted of 9 triplets of metacentric chromosomes. Fluorescence in situ hybridization (FISH) with the 18S rDNA probe clearly demonstrated the presence of 3 clusters of hybridization signals on the triplet of chromosome 7, thus confirming the triploid status of the specimens. FISH with a telomeric (TTAGGG)n probe confined hybridization signals exclusively to the terminal chromosomal regions, supporting the earlier findings that this repetitive motif is a conserved feature of tapeworm telomeres.

Morphological abnormalities in fish parasites: a potential tool for biomonitoring natural contaminants?

A comparative study on the strobilar morphology of the tapeworm Proteocephalus percae (Müller, 1780) (Cestoda), a parasite of the perch Perca fluviatilis (L.), showed a high percentage of abnormally developed parasite individuals. The evaluation of biological samples showed seven types of morphological abnormalities, mostly related to reproductive organs of the model tapeworm species. The most commonly identified deformity was an incomplete segmentation of the strobila. A malformed ovary, which is a structural anomaly linked with proglottization and maturation of the strobila, was also shown to be rather frequent. Offish hosts (P. percae) were collected from two localities with different levels of heavy metal pollution, the highly contaminated water reservoir Ruzín and a control locality, the water reservoir Palcmanská Masa, which belongs to the European network of protected areas in Slovakia. Tapeworm abnormalities occurred more frequently in individuals from the contaminated environment (29.9%) compared with individuals from the control site (4.9%). The concentrations of heavy metals found in the parasites and their fish hosts from the heavily polluted reservoir support our assumption that the occurrence of abnormalities could be linked with the destructive effect of toxic substances. The present study also demonstrates that the enumeration of body deformities exceeding the common level of phenotypic variability of particular parasitic species could potentially be used as an indicator of environmental problems.

Confirmation of the presence of zoonotic Trichobilharzia franki following a human cercarial dermatitis outbreak in recreational water in Slovakia.

Human cercarial dermatitis is a parasitic disease that causes an allergic reaction in the skin (swimmer's itch) as a consequence of contact with cercariae of bird schistosomes present in water, mainly of the genus Trichobilharzia Skrjabin et Zakarow, 1920. The main objective of the study was to confirm the presence of the zoonotic disease agent following reports of human infections in recreational water in Slovakia. We identified two species of freshwater snails at Kosice Lake, Radix auricularia (Linnaeus, 1758) and Physa acuta (Draparnaud, 1805). Trematode infections were observed only in R. auricularia. Of the 62 snails collected, 11 (17.7%) were infected with 5 different species of larval stages of trematodes. The blood fluke Trichobilharzia franki was found in 2 (3.2%) of the examined snails. The present record provides the first evidence that T. franki from the pulmonate snail R. auricularia represents a source of human cercarial dermatitis in recreational water in Slovakia. Our finding complements the easternmost records of both swimmer's itch and the confirmed occurrence of a bird schistosome in a waterbody in Europe. The present work suggests that the health risks associated with trichobilharziasis need to be further studied by detailed monitoring of the occurrence of the major causative agent of human cercarial dermatitis, T. franki.

Chromosomal study of Khawia abbottinae (Cestoda: Caryophyllidea): karyotype and localization of telomeric and ribosomal sequences after fluorescence in situ hybridization (FISH).

An original cytogenetic study combining classical karyotype analysis and modern fluorescence in situ hybridization using telomeric (TTAGGG)n and ribosomal sequences (18S rDNA) was performed in Khawia abbottinae (Cestoda, Caryophyllidea), a parasite of Chinese false gudgeon (Abbottina rivularis) from China. Analyses based on conventional Giemsa staining, DAPI, YOYO-1 dye, and silver (Ag) staining were also carried out. The karyotype is composed of eight pairs of metacentric and telocentric chromosomes (2n = 16, n=5m + 3t). Constitutive heterochromatin was mainly positioned at pericentromeric regions, and telomeric sequences (TTAGGG)n were restricted to the end of all chromosomes. In mitotic preparations stained with Giemsa, both homologues of chromosome pair 4 showed a distinct secondary constriction. FISH with rDNA probe confirmed that this secondary constriction contains a nucleolar organizer region (NOR). The process of spermatocyte meiosis and the dynamics of nucleolus degradation in dividing cell were scrutinized. The present study and its results enhance the limited knowledge on basic karyotype characteristics and 18S rDNA clusters location in caryophyllidean tapeworms.

Tapeworm chromosomes: their value in systematics with instructions for cytogenetic study.

The history and value of cytogenetic features for addressing questions of the evolution and systematics of tapeworms (Cestoda) are briefly reviewed along with instructions for collecting karyological data. As a supplement to worm morphology, chromosome number and morphology have been helpful in determining the systematic status of some genera in the Diphyllobothriidae and species in the Bothriocephallidea. In addition, many new techniques for chromosome analysis have been recently applied in morphological and molecular studies of invertebrates, including tapeworms. Methods of molecular karyology, fluorescence in situ hybridisation, and chromosomal location of satellite DNA, microsatellites or histone genes may also provide useful data to inference of taxonomic relationships and for revealing trends or general lines of chromosome evolution. However, as karyological data are available only for few tapeworms, they are seldom an integral part of evolutionary and taxonomic studies of cestodes. A primary reason for this lack of karyological data may lie in general difficulties in working with tapeworm chromosomes. To address these problems, herein we present a well-tested, step-by-step illustrated guide on the fixation of tapeworm material and preparation of their chromosomes for cytogenetic studies. The technique requires standard glassware, few reagents and simple equipment such as needles; it can also be used on other neodermatan flatworms.

Chromosome analysis and the occurrence of B chromosomes in fish parasite Acanthocephalus anguillae (Palaeacanthocephala: Echinorhynchida).

The cytogenetics of Acanthocephala is a neglected area in the study of this group of endoparasites. Chromosome number and/or karyotypes are known for only 12 of the 1,270 described species, and molecular cytogenetic data are limited to rDNA mapping in two species. The standard karyological technique and mapping of 18S rRNA and H3 histone genes on the chromosomes of Acanthocephalus anguillae individuals from three populations, one of which originated from the unfavorable environmental conditions of the Zemplínska Sírava reservoir in eastern Slovakia, were applied for the first time. All specimens had 2n = 7/8 (male/female); n = 1m + 1m-sm + 1a + 1a (X). Fluorescence in situ hybridization (FISH) revealed three loci of 18S rDNA on two autosomes and dispersion of H3 histone genes on all autosomes and the X chromosome. In addition to the standard A chromosome set, 34% of specimens from Zemplínska Sírava possessed a small acrocentric B chromosome, which was always found to be univalent, with no pairing observed between the B chromosome and the A complement. The B chromosome had a small amount of heterochromatin in the centromeric and telomeric regions of the chromosomal arms and showed two clusters of H3 genes. It is well known that an environment permanently polluted with chemicals leads to an increased incidence of chromosomal rearrangements. As a possible scenario for the B chromosome origin, we propose chromosomal breaks due to the mutagenic effect of pollutants in the aquatic environment. The results are discussed in comparison with previous chromosome data from Echinorhynchida species.

Title: Analyse chromosomique et présence de chromosomes B chez le parasite de poisson Acanthocephalus anguillae (Palaeacanthocephala, Echinorhynchida). Abstract: La cytogénétique des Acanthocephala est un domaine négligé dans l'étude de ce groupe d'endoparasites. Le nombre de chromosomes et/ou les caryotypes ne sont connus que pour 12 des 1270 espèces décrites, et les données cytogénétiques moléculaires se limitent à la cartographie de l'ADNr chez deux espèces. La technique caryologique standard et la cartographie des gènes de l'ARNr 18S et de l'histone H3 ont été appliquées pour la première fois sur les chromosomes d'individus d'Acanthocephalus anguillae provenant de trois populations, dont l'une dans les conditions environnementales défavorables du réservoir de Zemplínska Sírava dans l'est de la Slovaquie. Tous les spécimens avaient 2n = 7/8 (mâle/femelle); n = 1m + 1m-sm + 1a + 1a (X). La technique FISH a révélé trois locus d'ADNr 18S sur deux autosomes et une dispersion des gènes de l'histone H3 sur tous les autosomes et sur le chromosome X. En plus de l'ensemble standard de chromosomes A, 34 % des spécimens de Zemplínska Sírava possédaient un petit chromosome B acrocentrique, qui s'est toujours révélé univalent, sans aucun appariement observé entre le chromosome B et le complément A. Le chromosome B avait une petite quantité d'hétérochromatine dans les régions centromériques et télomériques des bras chromosomiques et présentait deux groupes de gènes H3. Il est bien connu qu'un environnement pollué en permanence par des produits chimiques entraîne une incidence accrue de réarrangements chromosomiques. Comme scénario possible pour l'origine du chromosome B, nous proposons des cassures chromosomiques dues à l'effet mutagène des polluants du milieu aquatique. Les résultats sont discutés en comparaison avec les données chromosomiques précédentes des espèces d'Echinorhynchida.

Acanthocephalans of marine and freshwater fishes from Taiwan with description of a new species.

During an ichthyoparasitological survey in 2017-2019, six species of acanthocephalans were found among Taiwan's freshwater (Cypriniformes: Xenocyprididae, Cyprinidae) and marine fishes (Scombriformes: Scombridae, Trichiuridae; Anabantiformes: Channidae; Carangaria/misc: Latidae): Micracanthorhynchina dakusuiensis (Harada, 1938), Rhadinorhynchus laterospinosus Amin, Heckmann et Ha, 2011, Pallisentis rexus Wongkham et Whitfield, 1999, Longicollum sp., Bolbosoma vasculosum (Rudolphi, 1819), and one new species, Micracanthorynchina brevelemniscus sp. n. All species are morphologically characterised and illustrated using light and scanning electron microscopy. The finding of R. laterospinosus, P. rexus and B. vasculosum is the first record for these species in Taiwan. Micracanthorhynchina brevelemniscus is similar to Micracanthorhynchina motomurai (Harada, 1935) and M. dakusuiensis in proboscis armature but differs from M. motomurai by larger eggs (53-59 × 15-16 µm vs 40 × 16 µm) and by the number of cement glands (6 vs 4) and from M. dakusuiensis by shorter body length (2.2-2.9 mm vs 4.0 mm in males and 2.9-4.1 mm vs 7.6 mm in females), by the location of the organs of the male reproductive system (from level of the posterior third of the proboscis receptacle in M. brevelemniscus vs in the posterior half of the trunk in M. dakusuiensis), and by length of lemnisci (lemnisci shorter than the proboscis receptacle vs lemnisci longer than the proboscis receptacle). Phylogenetic analyses of almost complete 18S rRNA gene revealed paraphyly of the family Rhadinorhynchidae suggested in previous studies. Micracanthorhynchina dakusuiensis and M. brevelemniscus formed a strongly supported cluster, which formed the earliest diverging branch to the rest of the rhadinorhynchids and transvenids.

Human diphyllobothriosis in Taiwan: A review of cases and molecular evidence of Dibothriocephalus nihonkaiensis.

Diphyllobothriosis is an infectious disease caused by the consumption of raw freshwater or marine fish containing larvae of broad tapeworms (Diphyllobothriidae). In the present study, we critically reviewed all cases of human diphyllobothriosis reported from Taiwan, including unpublished reports from hospitals in Taipei. Genotyping based on mitochondrial DNA marker (cox1) confirmed that two of the recent cases were caused by Dibothriocephalus nihonkaiensis, which is not native to Taiwan and was probably imported with Pacific salmon infected with larvae of D. nihonkaiensis. The causative species previously reported in Taiwan could not be definitively confirmed. However, considering the distribution of Dibothriocephalus latus, which is not endemic in Taiwan, past cases diagnosed as D. latus are questionable.

Hooking the scientific community on thorny-headed worms: interesting and exciting facts, knowledge gaps and perspectives for research directions on Acanthocephala.

Although interest in Acanthocephala seems to have reached only a small community of researchers worldwide, we show in this opinion article that this group of parasites is composed of excellent model organisms for studying key questions in parasite molecular biology and cytogenetics, evolutionary ecology, and ecotoxicology. Their shared ancestry with free-living rotifers makes them an ideal group to explore the origins of the parasitic lifestyle and evolutionary drivers of host shifts and environmental transitions. They also provide useful features in the quest to decipher the proximate mechanisms of parasite-induced phenotypic alterations and better understand the evolution of behavioral manipulation. From an applied perspective, acanthocephalans' ability to accumulate contaminants offers useful opportunities to monitor the impacts - and evaluate the possible mitigation - of anthropogenic pollutants on aquatic fauna and develop the environmental parasitology framework. However, exploring these exciting research avenues will require connecting fragmentary knowledge by enlarging the taxonomic coverage of molecular and phenotypic data. In this opinion paper, we highlight the needs and opportunities of research on Acanthocephala in three main directions: (i) integrative taxonomy (including non-molecular tools) and phylogeny-based comparative analysis; (ii) ecology and evolution of life cycles, transmission strategies and host ranges; and (iii) environmental issues related to global changes, including ecotoxicology. In each section, the most promising ideas and developments are presented based on selected case studies, with the goal that the present and future generations of parasitologists further explore and increase knowledge of Acanthocephala.

Title: Accrocher la communauté scientifique à des vers à la tête pleine d'épines : faits intéressants et passionnants, lacunes dans les connaissances et perspectives pour des orientations de recherche sur les Acanthocéphales. Abstract: Bien que l'intérêt pour les acanthocéphales semble n'avoir atteint qu'un petit nombre de chercheurs dans le monde, nous montrons dans cet article que ce groupe de parasites est composé d'excellents organismes modèles pour étudier les questions en suspens en biologie moléculaire et cytogénétique, écologie évolutive et écotoxicologie. Leur ascendance partagée avec les rotifères en fait un groupe idéal pour explorer les origines du mode de vie parasitaire et les moteurs évolutifs des changements d'hôtes et des transitions environnementales. Ils présentent également des caractéristiques intéressantes pour l'étude des mécanismes proximaux sous-tendant les altérations phénotypiques induites par les parasites, et ainsi mieux comprendre l'évolution de la manipulation comportementale. D'un point de vue appliqué, la capacité des acanthocéphales à accumuler les contaminants offre des opportunités utiles pour surveiller les impacts - et évaluer les possibilités d'atténuation - des pollutions anthropiques sur la faune aquatique et développer le domaine de la parasitologie environnementale. Cependant, l'exploration de ces pistes de recherche passionnantes nécessitera de relier des connaissances fragmentaires en élargissant la couverture taxonomique des données moléculaires et phénotypiques. Dans cet article, nous présentons l'état actuel de la recherche sur les acanthocéphales selon trois axes principaux : (i) la taxonomie intégrative (y compris les outils non-moléculaires) et la phylogénie à des fins d'analyse comparative ; (ii) l'écologie et l'évolution des cycles de vie, des stratégies d'exploitation des hôtes et de transmission ; (iii) les questions environnementales liées aux changements globaux, y compris l'écotoxicologie. Dans chaque section, nous soulignons les besoins et les opportunités, en espérant que cela incitera une nouvelle génération de parasitologues à s'intéresser aux acanthocéphales.

Classical and molecular cytogenetics of Khawia sinensis (Cestoda: Caryophyllidea), invasive parasite of carp, Cyprinus carpio.

Chromosomes of the invasive tapeworm Khawia sinensis (Caryophyllidea), the specific parasite of common carp, were analyzed by means of conventional Giemsa staining and using fluorescent DAPI and YOYO-1 dyes, silver staining, and fluorescent in situ hybridization (FISH) with 18S rDNA probe. The karyotype is composed of eight pairs of metacentric and telocentric chromosomes (2n = 16, n = 3m + 5t, TCL = 42.54 µm). Constitutive heterochromatin was located at pericentromeric regions of all pairs, except for the largest metacentric pair (no. 1), which possessed no DAPI-positive band. FISH with rDNA probe revealed that both homologues of chromosome pair no. 6 carry a cluster of ribosomal arrays, which were located interstitially close to the centromere. Present results are compared with previous cytogenetic data on Khawia spp., and comments are made on the karyotypes with respect to their phylogenetic links.

Spermiogenesis and spermatozoon ultrastructure of Hunterella nodulosa (Cestoda: Caryophyllidea), a monozoic parasite of suckers (Catostomidae) in North America.

Spermiogenesis and ultrastructure of mature spermatozoon of the caryophyllidean cestode Hunterella nodulosa, a parasite of suckers (Catostomidae), have been studied by transmission electron microscopy. This monozoic tapeworm is unique in its mode of attachment and represents the second North American species studied. The process of spermiogenesis of H. nodulosa follows the general pattern already described in other caryophyllideans. The most characteristic feature is the presence of a slight rotation of the flagellar bud, which seems to be a typical character of spermiogenesis in this cestode group. The mature spermatozoon of H. nodulosa is characterized by the presence of one axoneme of 9 + "1" type of the trepaxonematan flatworms surrounded by a semi-arc of cortical microtubules in its anterior extremity, parallel nucleus and cortical microtubules arranged in a parallel pattern, which corresponds to the Type III pattern of cestode spermatozoa according to Levron et al. (2010). Comparison of the present data with those available for other caryophyllideans did not reveal substantial differences, even though they belong to different families, infect different hosts (catostomid, cyprinid and siluriform fishes) and occur in distant zoogeographical regions. This indicates uniformity of the process of sperm formation and spermatozoon ultrastructure in one of the evolutionarily most ancient groups of tapeworms.

Comparative karyological analysis of four diplozoid species (Monogenea, Diplozoidae), gill parasites of cyprinid fishes.

The present study has revealed new data on chromosome complements of diplozoid parasites, namely Diplozoon paradoxum from freshwater bream Abramis brama, Paradiplozoon bliccae from white bream Blicca bjoerkna, Paradiplozoon sapae from white-eye bream Ballerus sapa, and Paradiplozoon nagibinae from zope Ballerus ballerus. Particularly, D. paradoxum is characterized by four pairs (2n=8) of long (up to 22.1 µm) chromosomes: pairs 1, 2, and 3 are metacentric and pair 4 acrocentric. Karyotypes of three Paradiplozoon species are nearly identical in number and morphological classification of chromosomes, each comprising diploid number of 14 one-armed chromosomes of very similar length ranging up to 12.5 µm in P. bliccae, 9.2 µm in P. sapae, and 9.9 µm in P. nagibinae. All four species are similar in their total complement length, ranging from 64.4 to 50.4 µm. Interspecific differences were found in location of secondary constriction: it is situated on short arm of the 1st chromosome pair in D. paradoxum, on long arm of the 7th pair in P. bliccae and on long arm of the 4th pairs in P. sapae and P. nagibinae. Phylogenetic interrelationship within the diplozoids and hypothetic karyotype evolution is here discussed.

Ultrastructure of spermiogenesis and mature spermatozoon of Breviscolex orientalis (Cestoda: Caryophyllidea).

Spermiogenesis and spermatozoon ultrastructure of the caryophyllidean cestode Breviscolex orientalis Kulakovskaya, 1962, first member of the family Capingentidae studied, a parasite of cyprinid fish Abbottina rivularis, are described using transmission electron microscopy. Spermiogenesis in B. orientalis follows the Type II pattern described by Bâ and Marchand (Mém Mus Natl Hist Nat 166:87-95, 1995) for cestodes. It begins with the formation of a zone of differentiation containing a large nucleus and a pair of centrioles. The centrioles are separated from one another by an intercentriolar body composed of three electron-dense layers. Each centriole is associated with typical striated roots. At the beginning of the spermiogenesis, an electron-dense material is observed in the apical region of the differentiation zone. During the initial stage of spermiogenesis, one of the centrioles gives rise to a free flagellum, which then rotates and undergoes proximodistal fusion with the cytoplasmic protrusion of the differentiation zone. The mature spermatozoon of B. orientalis corresponds to the Type III pattern described by Levron et al. (Biol Rev 85:523-543, 2010). It is characterized by the absence of mitochondrion and crested body. Five regions of the mature spermatozoon are differentiated. The main ultrastructural characteristics are: one axoneme of 9+ "1" trepaxonematan pattern, cortical microtubules and nucleus. The comparison of the spermiogenesis of B. orientalis with those of the other caryophyllidean species demonstrates some variation within the order relative to the presence and morphology of the intercentriolar body, the presence of slight rotation of the flagellar bud and a complete proximodistal fusion of the free flagellum with a cytoplasmic protrusion.

A comparative study of karyotypes and chromosomal location of rDNA genes in important liver flukes Fasciola hepatica and Fascioloides magna (Trematoda: Fasciolidae).

Chromosomal characteristics, i.e., number, size, morphology, and location of ribosomal DNA (rDNA) clusters were examined in two medically important liver flukes, Fasciola hepatica and Fascioloides magna (Fasciolidae), using conventional Giemsa staining and fluorescent in situ hybridization (FISH) with ribosomal 18S rDNA probe. A comparison of F. magna and F. hepatica karyotypes confirmed significant differences in all chromosomal features. Whilst the karyotype of F. hepatica comprised ten pairs of chromosomes (one metacentric and nine medium-sized subtelocentrics and submetacentrics; 2n = 20, n = 1 m + 5 sm + 4 st; TCL = 49.9 µm), the complement of F. magna was composed of 11 pairs of medium-sized subtelocentrics and submeta-metacentrics (2n = 22, n = 9 st + 1 sm + 1 sm-m; TCL = 35.2 µm). Noticeable differences were found mainly in length and morphology of first chromosome pair. It was metacentric and 9.0 µm long in F. hepatica while subtelocentric and 4.7 µm long in F. magna. Although FISH with rDNA probe revealed a single cluster of ribosomal genes in both species, conspicuous interspecific differences were displayed by chromosomal location of ribosomal loci (i.e., NORs). The signals were found on short arms of fifth homologous pair in F. hepatica; however, they were detected in pericentromeric regions of the long arms of tenth pair in F. magna. The observed cytogenetic differences were interpreted in terms of karyotype evolution of fasciolid flukes; F. hepatica may be regarded phylogenetically younger than F. magna. The present paper provides a pilot study on molecular cytogenetics within a group of hermaphroditic digenetic flukes.

Hazardous pollutants in the environment: Fish host-parasite interactions and bioaccumulation of polychlorinated biphenyls.

The present paper reports on the interrelationships of fish, parasites and the bioaccumulation of hazardous organic compounds in the Zemplínska Sírava water reservoir in eastern Slovakia, which is heavily polluted with polychlorinated biphenyls (PCBs). The concentrations of these contaminants were measured in various fish matrices (dorsal and abdominal muscle tissues, hepatopancreas, intestine wall and adipose tissue) of the freshwater bream, Abramis brama (Cyprinidae), and in its intestinal parasite Caryophyllaeus laticeps (Cestoda), which was used for the first time as a model for a PCB bioaccumulation study. Regarding the fish, the highest concentrations of PCBs were found in the intestine, followed by hepatopancreas and muscle tissues. The amounts of PCBs were higher in abdominal muscles than in their dorsal parts. Concentrations of ∑PCBs above the limits set by European regulations were detected in both muscle parts in the fish, confirming the persistent unfavorable conditions in this locality and high risk for biota and humans. Based on bioconcentration factor values (BCFs), PCBs reached much higher levels in cestodes compared to bream matrices. Some significant differences in PCB amounts between infected and uninfected bream were determined. Fulton's condition factor (CF) significantly differed in infected and non-infected fish (p ˂ 0.05), with CF values surprisingly lower in fish free of parasites compared to parasitized fish, which suggests a "mutualistic" relationship between the parasite and its host.

Hidden diversity of the most basal tapeworms (Cestoda, Gyrocotylidea), the enigmatic parasites of holocephalans (Chimaeriformes).

Gyrocotylideans are evolutionary ancient parasitic flatworms, and like their hosts-a relict group of holocephalan fishes (Chimaeriformes)-they are considered to be "living fossils" of a vanished past. However, the species diversity, host associations and biogeography of these most basal tapeworms are poorly known. Herein, we provide evidence of a conspicuous contrast between the genetic and morphological data based on an examination of newly collected and properly processed Gyrocotyle specimens (hologenophores) isolated from holocephalans off Taiwan and Argentina. Our molecular data, inferred from three genes (COI, 28S rRNA, 18S rRNA), showed unexpected genetic interrelationships among isolates of the genus Gyrocotyle, because each of the four genotypes from Taiwan clustered with isolates of distinct gyrocotylideans from the North Atlantic. Three genotypes of Gyrocotyle from Taiwan were morphologically almost indistinguishable from each other but represented distinct genetic lineages; a single specimen of Gyrocotyle sp. genotype 4 exhibited a clear genetic and morphological distinctness, though its formal description as a new species would be premature. Additionally, specimens of Gyrocotyle rugosa Diesing, 1850, from the type host Callorhinchus callorynchus from Argentina, provided the first genetic data on the type species of the genus and enabled us to characterise it, which is necessary for future taxonomic studies. The finding of some specimens of Gyrocotyle sp. genotype 3 in Chimaera phantasma, and another one in C. cf. argiloba, together with the putative conspecificity of an unidentified gyrocotylidean from Callorhinchus milii off Australia and G. rugosa from C. callorynchus off Argentina, represent evidence that one gyrocotylidean species may parasitise more than one holocephalan host species. Existing taxonomic problems and conflicts between morphological and molecular data on species of Gyrocotyle can only be resolved if hologenophores from type hosts and localities of nominal taxa are properly characterised genetically and morphologically.

A chromosome study and localization of 18S rDNA in Khawia saurogobii (Cestoda: Caryophyllidea).

This paper reports results of the first cytogenetic study carried out on a recently described monozoic tapeworm, Khawia saurogobii Xi et al., 2009, from the Chinese lizard gudgeon (Saurogobio dabryi). The karyotype of this species is composed of eight pairs of metacentric and telocentric chromosomes (2n = 16; n = 3m + 5t), metacentric chromosomes representing the first, sixth, and eight pairs. All chromosomes except the largest pair displayed 4',6-diamidino-2-phenylidole (DAPI) positive heterochromatin in centromeric regions. In mitotic preparations stained with Giemsa, one of the homologues of a smaller metacentric chromosome pair (No. 7) showed a distinct secondary constriction, whereas the other did not. Fluorescent in situ hybridization (FISH) with 18S ribosomal DNA (rDNA) probe revealed that the chromosomes No. 7 carry each a cluster of ribosomal genes associated with the centromeric heterochromatin and confirmed that this chromosome pair contains a nucleolar organizer region (NOR). The rDNA-FISH also confirmed heteromorphism in the size of NOR (i.e., secondary constriction) observed after Giemsa staining. The present cytogenetic analysis revealed species-specific characters of K. saurogobii and showed that FISH may represent a new valuable cytogenetic tool suitable for comparative taxonomic or phylogenetic studies within the order Caryophyllidea in the future.

Effect of fungicide Euparen Multi (Tolylfluanid) on the induction of chromosomal aberations in cultivated bovine lymphocytes.

The effect of the fungicide Euparen Multi (containing 50% tolylfluanid) was investigated on the induction of chromosomal aberrations (CA) in cultured bovine peripheral lymphocytes. Cultures from two healthy donors were treated with tolylfluanid-based fungicide at concentrations ranging from 1.7 to 17.5 µg/ml for the last 24 and 48 hours of cultivation. Conventional cytogenetic method (CA assay) with Giemsa staining as well as fluorescence in situ hybridization (FISH) with whole bovine chromosomes 1 and 5 painting probes were used in the experiment. In the CA assay, no clastogenic effect of the fungicide was found after Euparen Multi treatment for 24 hours. On the contrary, significant elevation in polyploidy induction was observed with dose-dependence in one of the donors. Using prolonged time of exposure to the fungicide (the last 48 h of the cultivation), a slight clastogenic effect was detected at the doses of 8.75 and 17.5 µg/ml (P < 0.05, P < 0.01, respectively) in donor 1 and at the dose of 8.75 µg/ml (P < 0.05) in donor 2. The highest doses tested caused reduction of the mitotic indices (MI) (P < 0.05, P < 0.01) in both donors as well as both treatment times. The evaluation of stable structural aberrations in lymphocytes by two-colour FISH (48 h exposure) using bovine chromosome painting probes revealed the presence of nonreciprocal translocations at two examined concentrations (3.5 µg/ml and 8.75 µg/ml).

Cytogenetics of Eudiplozoon nipponicum (Monogenea, Diplozoidae): Karyotype, spermatocyte division and 18S rDNA location.

Ectoparasitic monogeneans of the family Diplozoidae have direct and monoxenous life cycle. The cytogenetics of monogeneans in general and diplozoids in particular, is a relatively underexplored area. This is why each new detailed description of a karyotype provides significant information about the evolution of monogenean chromosomes and contributes to a better understanding of phylogenetic relationships within this group. This study offers new data on the chromosomes of Eudiplozoon nipponicum, an invasive parasite of the common carp. This species' karyotype consists of seven pairs of telocentric chromosomes (2n = 14 t). After DAPI staining, we marked heterochromatin blocks on all chromosomes in the pericentromeric region. Silver staining (AgNO3) and staining with fluorescent dye YOYO-1 revealed the presence of one large active nucleolus. Fluorescent in situ hybridization with an 18S rDNA probe revealed one cluster of ribosomal genes at the terminal part of the long arms of chromosome pair No. 7. We compared our results with studies on the phylogenetic relationships of diplozoids which applied a combination of molecular methods and classical morphological characterization and found that the results of our cytogenetic analysis are consistent with the hypothesis that E. nipponicum is more basal member of the family Diplozoidae.