Redescription of Paradiplozoon opsariichthydis (Jiang, Wu et Wang 1984) Jiang, Wu et Wang, 1989 (Monogenea, Diplozoidae).

Paradiplozoon opsariichthydis (Jiang, Wu et Wang, 1984) Jiang, Wu et Wang, 1989 (Platyhelminthes, Monogenea, Diplozoidae) is blood-feeding parasite from the gills of Asian cyprinid fish Opsariichthys bidens Günther, 1873. In this study, we present a morphological redescription of P. opsariichthydis neotype main morphological features e.g. size of body and clamps due to the fact that the type material is missing. We decided to supplement morphological descriptions by the relevant molecular data (internal transcribed spacer - ITS2) related to P. opsariichthydis adult worm isolates and other representatives of genus Paradiplozoon to cross verify our findings. In addition to that, this study also brings an attention to the host identification. Thus, parasite data were complemented by the determinant cytochrome oxidase b (cytb) sequences of its hosts. All novel sequences are deposited in GenBank. This combination of the morphological and molecular data related to both the parasite and its host seems to be the optimal approach to the general process of (re)description of highly host-specific parasitic organisms, which can then lead to a meaningful phylogenetic analysis.

Diversity and phylogeny of Paradiplozoon species (Monogenea: Diplozoidae) parasitising endemic cyprinoids in the peri-Mediterranean area, with a description of three new Paradiplozoon species.

The Diplozoidae are monogenean parasites of mainly cyprinoid fishes with a unique life cycle, whereby two larvae undergo anastomosis and fuse into a single cross-shaped specimen. Paradiplozoon is the most species rich and widespread genus of the family, with a distribution range covering Eurasia and Africa; however, some areas remain underexplored and their diplozoid fauna is uncertain. In the present study, the Paradiplozoon diversity was investigated in the peri-Mediterranean region, which has the highest levels of cyprinoid diversity and endemism in Europe. A total of 36 endemic cyprinoid species were sampled from sites in north-west Africa and the southern European peninsulas and investigated for the presence of diplozoid parasites. Of five Paradiplozoon species collected, three were identified as new to science: Paradiplozoon moroccoensis n. sp. from the Moroccan endemic Luciobarbus lepineyi; Paradiplozoon ibericus n. sp. from Iberian endemic cyprinids and leuciscids and Paradiplozoon helleni n. sp. from the Greek endemic Scardinius acarnicus and Tropidophoxinellus hellenicus (descriptions provided herein). In addition, new host records for P. homoion and P. megan are presented, with the former being most prevalent in the investigated region. Phylogenetic analysis supported paraphyly of the genus Paradiplozoon, and suggests the need for a careful taxonomic re-evaluation of this genus. Furthermore, the results showed that endemic Paradiplozoon of the peri-Mediterranean do not form a monophyletic group, suggesting multiple origins of this parasite groups in different peri-Mediterranean regions.

Redescription of Paradiplozoon hemiculteri (Monogenea, Diplozoidae) from the type host Hemiculter leucisculus, with neotype designation.

Paradiplozoon hemiculteri (Ling, 1973), a member of the Diplozoidae, parasitizes the gills of Asian fish. Not only is the type material unavailable for this species, the original description was poor and somewhat conflicting, and adequate molecular data were not available. What is more, the available morphological and molecular data are inconsistent and fluctuate significantly. Here, we present a redescription of P. hemiculteri based on morphological and molecular data from new isolates collected from the type host, the sharpbelly Hemiculter leucisculus (Basilewsky, 1855), captured at the neotype locality (Shaoguan, Guangdong Province, southern China); a neotype for P. hemiculteri was designated from this collection. The length and width of the body, buccal suckers, pharynx, attachment clamps, sickle and the central hook handle were all measured and the shape of the anterior and posterior part of the median plate and anterior and posterior joining sclerites accurately documented. Phylogenetic analyses based on the sequences of the second rDNA internal transcribed spacer (ITS2) indicated that all new samples clustered together and differed clearly from sequences attributed to P. hemiculteri, which are deposited in GenBank. Our results confirm that P. hemiculteri is the only diplozoid that has demonstrably been found on the gills of H. leucisculus to date.

Morphological and Molecular Assessment of Pentastomes from Gulls in Portugal.

During 2016, 153 gulls, including 64 Larus fuscus and 89 Larus michahellis, were found crippled in south Portugal. They died in Wildlife Rehabilitation and Investigation Center-RIAS and were necropsied. Reighardia sternae infected 2 (3%, n = 64) L. fuscus and 4 (4%, n = 89) L. michahellis. Molecular analysis confirmed the morphological identification on the basis of total body length, maximum body width, length of anterior and posterior hooks, total length of oral apparatus, and other features of oral apparatus of R. sternae. Both sequenced individuals in this study displayed 100% sequence identity to R. sternae individuals obtained previously from Larus ridibundus in Spain and to Reighardia sp. from Larus belcheri in Peru. Reighardia sternae is reported here for the first time in L. michahellis and for the first time from Portugal.

An ultrastructural study of the surface and attachment structures of Paradiplozoon homoion (Bychowsky & Nagibina, 1959) (Monogenea: Diplozoidae).

BACKGROUND: Species of Diplozoon Palombi, 1949 (Monogenea: Diplozoidae) are blood-feeding ectoparasites mainly parasitising the gills of cyprinid fishes. Although these parasites have been the subject of numerous taxonomic, phylogenetic and ecological studies, the ultrastructure of the surface and haptor attachment structures remains almost unknown. In this study, we used transmission electron microscopy to examine the ultrastructure of attachment clamps and neodermal surface of Paradiplozoon homoion (Bychowsky & Nagibina, 1959), family Diplozoidae Palombi, 1949, thereby broadening our knowledge of platyhelminth biology. RESULTS: The hindbody surface of P. homoion is distinctly ridged, each ridge being supported by several muscle fibers and equipped with scales on the surface plasma membrane. Such structures have not been recorded previously in species of the family Diplozoidae. Comparisons of the surface structure of different body parts revealed slight differences in the thickness and number of organelles. Each of the clamps has a flattened bowl-like structure composed of sclerites, movable skeletal-like structures that are anchored by robust, radially oriented muscle bundles. The base of the posterior median plate sclerites is equipped with glandular cells possessing secretory vesicles. CONCLUSION: This study brings detailed ultrastructural data for the surface and haptoral attachment clamps of P. homoion and provides new insights into the ultrastructure of Diplozoidae. Glandular cells at the base of the attachment clamps responsible for sclerite development in diplozoid species were observed for the first time. Our findings support the hypothesis that the structure of particular neodermal compartments is similar within the Platyhelminthes. On the other hand, the diplozoid glandular system and the mechanism of sclerite development clearly merits further attention.

New data on the karyotype and chromosomal rDNA location in Paradiplozoon megan (Monogenea, Diplozoidae), gill parasite of chubs.

New morphological data on chromosome complement of diplozoid parasite Paradiplozoon megan from the chub Squalius cephalus are shown herein. The karyotype of P. megan is characterized by seven pairs (2n = 14) of medium long (up to 11 µm) one-armed chromosomes which are nearly identical in number and morphological classification with chromosomes of other Paradiplozoon species described karyologically to date (Paradiplozoon bliccae, Paradiplozoon nagibinae, Paradiplozoon sapae, Paradiplozoon pavlovskii and Paradiplozoon homoion). A single locus for ribosomal RNA genes, visualized in the secondary constriction site by the fluorescent in situ hybridization method, is situated interstitially on a median part of a long arm of the smallest, 7th chromosome pair in P. megan. Phylogenetic interrelationships within the members of the family Diplozoidae and hypothesis for the ancestral karyotype are discussed here.

The molecular and morphometrical description of a new diplozoid species from the gills of the Garra rufa (Heckel, 1843) (Cyprinidae) from Turkey--including a commentary on taxonomic division of Diplozoidae.

The paper presents a description of Paradiplozoon bingolensis sp. n. from the gills of Garra rufa Heckel, 1843 (Cyprinidae) collected from the Göynük Stream, a tributary of the Murat River, Turkey. This is the first diplozoid species to be described from G. rufa. P. bingolensis is distinguished from the other valid species in the genus by the combination of the morphology of the sclerites of its clamps and by the size of the central hooks. Even molecular characterization based on variability of the second internal transcribed spacer (ITS2) of the ribosomal DNA region provided additional support of separation of this new species from the valid ones. The sequences were compared with previously published ITS2 sequences of other diplozoid species. Subsequent analysis demonstrated the uniqueness of this new parasite species and revealed uncertainties in the current taxonomic division of the Diplozoidae that are commented in the text.

Redescription and resurrection of Bertiella satyri (Cestoda, Anoplocephalidae) parasitizing the orangutan (Pongo abelii) in Indonesia.

The tapeworm species Bertiella satyri from a semi-wild Sumatran orangutan (Pongo abelii: Ponginae) is redescribed and the sequence of its 18S rDNA is presented. The tapeworms parasitizing the genera Pan, Pongo, Homo and Hylobates from Muséum National d'Histoire Naturelle, Paris and from Muséum d´Histoire Naturelle, Genève are also presented. The validity of B. satyri is confirmed. B. satyri (BSA) differs from the most similar species Bertiella studeri (BSTU) in the following characteristics: (1) testes number, BSTU 300-400; BSA 116-124, (2) genital opening, BSTU regularly alternate; BSA irregularly alternate, (3) Cirrus-sac, BSTU short, 0.250-0.320, does not reach excretory ducts; BSA long, 0.630 × 0.495, reaches excretory ducts, (4) egg size, BSTU 0.053-0.060; BSA 0.030-0.051, (5) host BSTU Pan troglodytes, Africa; BSA Pongo pygmaeus, P. abelii, Indonesia (Borneo, Sumatra). Both species differ according to our material and the name B. satyri (Blanchard, 1891) is resurrected.

Eudiplozoon nipponicum in focus: monogenean exhibiting a highly specialized adaptation for ectoparasitic lifestyle.

Developmental stages of the diplozoid monogenean Eudiplozoon nipponicum, comprising oncomiracidium, diporpa, juvenile, and adult, were investigated using light and scanning electron microscopy in conjunction with confocal scanning laser microscopy in order to examine body organization and identify explicit morphological adaptations to the ectoparasitic life in each stage. The parasite exhibits a complex digestive tract well equipped for hematophagous feeding. It consists of a mouth opening with prominent buccal suckers, eversible pharynx with adjacent glandular structures, and a blind-ending gut with cecal lining. Glandulo-muscular organs, located apically and opened into the mouth corner, are considered to be a part of the digestive tract. Based on our observations of pharynx eversion and in light of the presence of several glandular or gland-like structures, we propose a new hypothesis on the possibility of extracorporeal digestion of this parasite. The hindbody bears an attachment apparatus, comprising haptor, lobular extensions, and tegumental folds, responsible for the parasite's firm attachment to the host gills. The possibility of buccal suckers assisting in the parasite's translocation while searching for an optimal niche or their temporary attachment function during feeding is discussed. The body of each compound adult (i.e., permanent copula) is almost completely filled by two complete reproductive tracts comprising the female as well as male organs. Such a reproductive strategy, in which two independent heterogenic individuals fuse into a single hermaphrodite organism without the need to search for mating partner, represents a high specialization of diplozoids to their parasitic life.

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.

Ethanol-fixed material used for both classical and molecular identification purposes: Eudiplozoon nipponicum (Monogenea: Diplozoidae) as a case parasite species.

This study is focused on the feasibility of two treatments of alcohol-fixed monogenean parasites which are intended to be use for the combined morphological and molecular characterizations. The monogenean parasite, Eudiplozoon nipponicum, was selected as a model parasite species; however it is expected that these techniques will be suitable for other monogeneans and other parasitic families. The haptor of diplozoid parasites is equipped with sclerotized attachment clamps and central hooks which are utilized for morphological identification. As parasite tissue become very tough and rigid when preserved in ethanol, using these structures for species identification without additional treatment is difficult. We investigated two different techniques to digest the surrounding tissues, the first was treatment with 10% sodium dodecyl sulphate (SDS) and the second treatment was proteinase K. Tissue was successfully digested in both treatments and all clamps, central hook and even individual sclerites of the clamps were clearly visible and well defined. After treatment, the digest was used to extract genomic DNA, and the second internal transcribed spacer of the ribosomal DNA genes (rDNA) was amplified. Nucleic acid sequence was obtained from 90% of parasite specimens processed by both treatments. Treatment of haptors with SDS was proven to be more successful with no visible changes or damage observed to sclerites even after a month. This method represents a useful tool for the combined morphological and molecular studies as the correct sequence can be assigned to the same individual worm from which haptoral parts have been obtained.

Description of Lemuricola (Lemuricola) pongoi--male (Nematoda: Enterobiinae) parasitising orangutan Pongo abelii.

The study presents the first description of male specimen (allotype) of the pinworm species Lemuricola (Lemuricola) pongoi, parasitising Sumatran orangutan (Pongo abelii) from Sumatra (Indonesia). The male specimen morphology presents all features fully corresponding with diagnose of the genus and subgenus Lemuricola. We are agreeing that Protenterobius is synonymous with Lemuricola because the length of tail appendix in males is not a sufficient feature for separation of the genera or subgenera. For L. (L.) pongoi male are characteristic features: body length (4,803 microm), total oesophagus length (530), tail length (235), tip of tail length (188) and spicule length (90).

Telomere analysis of platyhelminths and acanthocephalans by FISH and Southern hybridization.

We examined the composition of telomeres in chromosomes of parasitic worms, representatives of the flatworm groups Monogenea and Cestoda (Platyhelminthes), and thorny-headed worms (Syndermata: Acanthocephala) by fluorescence in situ hybridization (FISH) with different telomeric repeat probes. Our results show that the (TTAGGG)n sequence, supposed to be the ancestral telomeric repeat motif of Metazoa, is conserved in Monogenea (Paradiplozoon homoion) and Cestoda (Caryophyllaeus laticeps, Caryophyllaeides fennica, and Nippotaenia mogurndae) but not in Acanthocephala (Pomphorhynchus laevis and Pomphorhynchus tereticollis). In the Pomphorhynchus species, no hybridization signals were obtained with the "nematode" (TTAGGC)n, "arthropod" (TTAGG)n, and bdelloid (TGTGGG)n telomeric probes using FISH with their chromosomes and Southern hybridization with P. laevis DNA. Therefore, we suggest that parasitic Acanthocephala have evolved yet unknown telomeric repeat motifs or different mechanisms of telomere maintenance.

Morphometric and molecular characteristics of Labeonema synodontisi n. comb. (Nematoda: Atractidae) from the West African fishes.

Numerous nematodes were found in the rectum of three fish species Synodontis ocellifer, S. nigrita, and S. schall (Mochokidae, Siluriformes) from the Gambia River and Mare Simenti, National Park Niokolo Koba, Senegal. A nematode species Raillietnema synodontisi Vassiliadès, 1973 (host S. ocellifer), is redescribed using morphometric (including scanning electron microscopy) and molecular characteristics and transferred into the genus Labeonema Puylaert, 1970. It is morphologically and metrically similar to Labeonema intermedium Puylaert, 1970, the other congeneric species (L. bainae Baker, 1982; L. bakeri Van Waerebeke, Chabaud, Bain et Georges, 1988; and L. africanum Moravec et Van As, 2004) differ from them either by the spicule and gubernaculum lengths, distribution and number of pre-cloacal papillae, position of the vulva, as well as hosts and geographical distribution. The partial sequences of small ribosomal subunit rDNA of L. synodontisi were analyzed and compared with other nematode sequences. Molecular analyses seem to support the position of this nematode species based on the morphological observation.

Investigation of Paradiplozoon homoion (Monogenea, Diplozoidae) life cycle under experimental conditions.

Diplozoids (Diplozoidae, Monogenea) are fish ectoparasites with a direct life cycle without intermediate hosts. Their free swimming larva, the oncomiracidium, hatches from eggs, invades a fish host and metamorphoses into a post-oncomiracidial larval stage, the diporpa. Later, two diporpae fuse and live as a pair in cross-copulation during their adult life. An experimental study was designed to investigate the life cycle of Paradiplozoon homoion (Monogenea, Diplozoidae) parasitizing their common fish hosts, gudgeon (Gobio gobio). A total of 35 gudgeon parasitized by diplozoids were collected from their natural environment of the Vlára River, Czech Republic, and kept together in tanks with 41 non-parasitized gudgeons reared in a laboratory environment. In total, 100 adult specimens of P. homoion were collected from the Vlára River gudgeon and a new parasite generation was expected to be observed on fish reared in the laboratory environment. Eight days after the first diplozoid eggs appeared on fish gills, the presence of diporpae with one or two pairs of clamps was noted. The appearance of the first juveniles was recorded at the same time as diporpae. Development of P. homoion from egg to sexually mature adult stage took 33 days at a constant temperature of 20 degrees C. The development of eggs in adults of the second generation was observed 2 days after the first observation of these adults. The behavior of oncomiracidia was also studied and this free swimming stage of diplozoids survived for 22 h in the absence of a host. When host fish were experimentally infected by oncomiracidia, diporpae were found attached to the fish gill apparatus within 2 h of infection.

A new species of Pterothominx Freitas, 1959 (Nematoda: Capillariidae) parasitising psittacine birds (Psittaciformes).

A new species, Pterothominx (Pterothominx) moraveci n. sp. (Trichinelloidea: Capillariidae) is described from an Australian parrot, Barnardius zonarius Shaw (Psittacidae: Psittaciformes), imported from Germany and kept in captivity in the Czech Republic. Males of the new taxon differ from all other species of the subgenus Pterothominx Freitas, 1959, which mainly parasitise galliform birds, in having a shorter spicule (0.925-1.338; mean 1.050 mm), a shorter body length and a different form of armed spicular sheath and bursa. Females differ in having the smallest eggs (40-50 x 20-25; mean 48 x 23 microm) and in other morphometrical characters. From the species P. (P.) totani (Linstow, 1875), which parasitises charadriiform birds, the new species differs in its smaller number of stichocytes (females 42-46, mean 44; versus 50-54), different form of the bursa and praebursal alae, and other characters. This is the only species of Pterothominx that parasitises psittacine birds.

Associations between fish reproductive cycle and the dynamics of metazoan parasite infection.

The parasite fauna of the stone loach (Barbatula barbatula) was investigated monthly from May to December 2001. Four parasite species were recorded: Gyrodactylus spp. (Monogenea), Tylodelphys clavata (Digenea), Proteocephalus sagittus (Cestoda), and Raphidascaris acus (Nematoda). The changes in the seasonal dynamics of parasite infection were investigated throughout the 8 months. The potential effect of the reproductive investment of the host, measured by gonad mass and gonado-somatic index (GSI), on the parasite infection was tested against the prediction that, during periods of high reproductive investment (beginning of the breeding period or forming gonads after breeding), the fish are more susceptible to parasite infection. Differences between parasite loads between genders were also hypothesized. Seasonal differences in infection were observed for all parasite species studied. The values of GSI showed a pattern of energy accumulation in the pre-reproductive period and at the beginning of breeding, a decrease during breeding, and an increase in the post-breeding period. A similar pattern was observed for parasite abundance, a strong or weak increase in spring and/or autumn and a decrease during summer (July and August). Positive correlations between the abundance of Gyrodactylus spp. and R. acus and both gonad mass and GSI were found in females after eliminating the effect of fish weight. Our results suggest that stone loach females are more susceptible to parasite infection in periods of higher reproductive investment. The main factor determining the infection of T. clavata was fish size. The abundance of P. sagittus was positively correlated with GSI in the total fish sample with no detectable effect of sex or fish weight. The increase in cestode infection in spring supports the hypothesis that the parasite life cycle could by synchronized with the beginning of host reproduction, probably induced by increasing fish hormone levels in the spring.

Development in vitro of the neuromusculature of two strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus.

Confocal microscopy interfaced with cytochemical procedures has been used to monitor development of the major muscle systems and associated serotoninergic (5-HT, 5-hydroxytryptamine) and peptidergic (FaRP, FMRFamide-related peptide) innervation of the strigeid trematodes, Apatemon cobitidis proterorhini and Cotylurus erraticus during cultivation in vitro. Sexually undifferentiated metacercariae were successfully grown to ovigerous adults using tissue culture medium NCTC 135, chicken serum and egg albumen. Eggs were produced after 5 days in culture but had abnormal shells and failed to embryonate. 5-HT and FaRP (the flatworm FaRP, GYIRFamide) were localised immunocytochemically in both central and peripheral nervous systems of developing worms. During cultivation, the central serotoninergic and FaRPergic neuronal pathways of the forebody became more extensive, but retained the same basic orthogonal arrangement as found in the excysted metacercaria. Longitudinal extensor and flexor muscles of the hindbody provide support for the developing reproductive complex. The male reproductive tracts were established in advance (day 3) of those of the female system (day 4); completion of the latter was marked by the appearance of the ootype/egg chamber. The inner longitudinal muscle fibres of the female tract appeared prior to the outer and more densely arranged circular muscles. Circular fibres dominate the muscle complement of both alimentary and reproductive tracts. 5-HT- and GYIRFamide-immunoreactivities were demonstrable in the central nervous system (CNS) and subtegumental parasympathetic nervous system (PNS) throughout the culture period, but innervation of the developing reproductive structures was reactive just for 5-HT. Only at the onset of egg production was FaRP-IR observed in the reproductive system and was expressed only in the innervation of the ootype, a finding consistent with the view that FaRPs may regulate egg assembly in platyhelminths.

Resurrection of Proteocephalus sagittus (Grimm, 1872) (Cestoda: Proteocephalidea) based on morphological and molecular data.

This paper presents a redescription of the proteocephalidean cestode Proteocephalus sagittus (Grimm, 1872) based on freshly collected specimens from the type-host, the stone loach Barbatula barbatula (L.) (family Balitoridae), from the Czech Republic. This species has recently been synonymised with P. torulosus (Batsch, 1786), a parasite of cyprinid fishes in the Holarctic Region, but it differs in the following characters: (i) scolex morphology (scolex rounded, with more apically situated suckers and lacking longitudinal wrinkles in P. sagittus versus large and club-shaped, with more laterally situated suckers and longitudinal wrinkles in P. torulosus); (ii) position of the vagina (opens anterior to the cirrus-sac in P. sagittus versus overlapping its distal region dorsally in P. torulosus); (iii) length of the cirrus-sac (shorter in P. sagittus); (iv) osmoregulatory canals (more distinct and situated more laterally, but almost indistinguishable and more medial in P. torulosus); and (v) sequences of the partial 18S rRNA (V4 region - 96.9% similarity) and 5.8S-ITS2 genes (65.9% similarity). P. pamirensis Dzhalilov & Ashurova, 1971, a poorly described species from the Tibetan stone loach Noemacheilus stoliczkai[= Tryplophysa stoliczkae (Steindachner)] in Tadjikistan, is synonymised with P. sagittus.

The first species of Thelastoma Leidy, 1849 (Nematoda: Thelastomatidae) parasitising the scorpion Euscorpius italicus (Chactidae: Scorpionidea).

A new species, Thelastoma euscorpii, (Thelastomatidae: Oxyuroidea) is described from Euscorpius italicus (Chactidae) on Pag Island (Kvarner Islands), Croatia. The new taxon differs from all other species of Thelastoma in having a cuticle with fine transverse striae. The excretory pore of the female specimens is located in a prebulbar position, the tail is short and subfiliform (5.5-9.3% of the total body length), and the eggs are small (47-52 x 24-29 microm). The males have a short oesophagus and no spicules. This is the first nematode species found in scorpions (Scorpionidea).