Karyological studies on three strigeid digeneans: Ichthyocotylurus erraticus (Rudolphi, 1809), I. variegatus (Creplin, 1825) and Apatemon gracilis (Rudolphi, 1819)

Previous work has shown that karyology may be a valuable technique for discriminating morphologically similar species. In the present study, chromosome number and morphology were described for three strigeid species, Ichthyocotylurus erraticus (Rudolphi, 1809), I. variegatus (Creplin, 1825) and Apatemon gracilis (Rudolphi, 1819). The results show that these species possess the same diploid number of chromosomes (2n = 20) in somatic cells. The relative length and the position of the centromere were determined, and significant inter-specific differences in the karyotypes are demonstrated, enabling the ready discrimination of I. erraticus and I. variegatus cercariae. A review of known karyotypes of the family Strigeidae suggests that possible routes of evolutionary changes involve pericentric inversions within chromosomes rather than Robertsonian translocations between elements.     

Multivariate analyses of morphometrical features from Apatemon gracilis (Rudolphi, 1819) Szidat, 1928 and A. annuligerum (v. Nordmann, 1832) (Digenea: Strigeidae) metacercariae

Only two species of the strigeid subgenus Apatemon (Apatemon) sensu Sudarikov (1959), Apatemon gracilis (Rudolhi, 1819) and A. annuligerum (von Nordmann, 1832), are known to infect British fishes. A. annuligerum is poorly characterised, with few life-cycle data available, since only the adult and metacercarial stages have been described. Both of these life-stages demonstrate only minor morphological differences from those of A. gracilis. The multidimensional approach of principal components analysis (PCA) was employed to determine the intra- and inter-specific morphological variation that exists for metacercariae of these species. Analyses revealed that metacercarial morphometrics cannot confidently discriminate between Apatemon gracilis and A. annuligerum specimens. Indeed, levels of intra-specific variation present between A. gracilis metacercariae originating from different piscine hosts were as marked for certain populations as those between the two nominal species. Individual populations of A. gracilis metacercariae differed most in the relative dimensions of their internal organs, whereas variation between the two nominal species was primarily in length-related variables. A. gracilis specimens originating from Welsh and Scottish stoneloach represented two populations with the most comparable total body lengths, yet clustered separately. Conversely, A. gracilis metacercariae infecting rainbow trout and salmon parr differed markedly in size, but demonstrated minimal composite morphological variation. Much, if not all, of the variation described between populations and species of parasites can be attributed to host or to metacercarial location within a particular host. These findings and the lack of strong life-cycle data for A. annuligerum place some doubt on its validity as a species discrete from A. gracilis.     

Molecular evidence for the synonymy of two species of Apatemon Szidat, 1928, A. gracilis (Rudolphi, 1819) and A. annuligerum (von Nordmann, 1832) (Digenea: Strigeidae) parasitic as metacercariae in British fishes

The current study examined rDNA (internal transcribed spacer regions, ITS1 and ITS2) and cytochrome c oxidase subunit 1 (CO1) sequence data of Apatemon annuligerum (originating from two geographical locations) and A. gracilis metacercariae (originating from four natural piscine hosts) to determine the systematic status of these two strigeid digeneans. With the exception of short repeat motifs, the ITS1 regions sequenced demonstrated no intra- or inter-specific sequence variation. ITS2 sequences were 292 bp and CO1 sequences 366 bp in length and identical for both nominal Apatemon species. These sequence data provide strong evidence that the two species are con-specific and that A. annuligerum should be regarded as a junior synonym of A. gracilis.     

Variation in the morphology of adult Apatemon gracilis Rudolphi, 1819 (Digenea: Strigeidae) reared in different avian hosts

Adult Apatemon gracilis (Rudolphi, 1819) were reared experimentally in three different avian hosts: herring gulls Larus argentatus Gmelin, domestic chicks Gallus gallus (L.) and eider ducklings Somateria mollissima (L.). Comparisons of size, body proportions and fecundity were made between these and specimens obtained from a naturally infected goosander Mergus merganser L. The eider duck proved to be a suitable experimental host, rearing adults of comparable size and fecundity to those from the natural host. The growth and development of the parasite in domestic chicks and herring gulls were significantly reduced. Similar conditions are likely to exist in the natural environment and this should be taken into consideration when choosing experimental hosts for use in the laboratory or when identifying species.     

A molecular phylogeny of the genus Ichthyocotylurus (Digenea, Strigeidae)

Three nucleotide data sets, two nuclear (ribosomal internal transcribed spacer regions 1 and 2, ITS1 and ITS2) and one mitochondrial (cytochrome c oxidase subunit 1, CO1), were analysed using distance matrix and maximum likelihood methods to determine the inter-relationships amongst the four species attributed to the genus Ichthyocotylurus Odening, 1969. Sequence data obtained from all gene loci investigated supported the position of Ichthyocotylurus variegatus as a species discrete from Ichthyocotylurus platycephalus. Phylogenetic analyses yielded congruent trees, with I. variegatus isolates comprising a common clade to which I. platycephalus constitutes a sister taxon. Ichthyocotylurus erraticus and Ichthyocotylurus pileatus were found to demonstrate a similarly close inter-specific relationship. The greatest intra-generic divergence occurred in the CO1 region (16% variability), with resultant disparities in three to eight encoded amino acids. PCR amplification yielded multiple ITS1 products for all Ichthyocotylurus spp. Analyses of equivalent-sized amplicons showed 5.4% intra-generic variation and several point mutations between I. variegatus isolates from different geographical localities and from different piscine hosts. The ITS2 locus was extremely conserved, with less than 1% variation between species. No intra-specific variation was recorded for any CO1 or ITS2 sequences.     

Chromosome studies of Ichthyocotylurus platycephalus (Creplin, 1825) Odening 1969 with description of triploid variant and comparative karyology of the genus Ichthyocotylurus

This paper reports the first karyological study of trematode Ichthyocotylurus platycephalus (Creplin, 1825) Odening 1969. Chromosome number and morphology were studied in somatic cells of parthenites from intermediate host--mollusc Valvata piscinalis using an air drying technique and Giemsa staining. The karyotype consisted of 20 chromosomes comprising five pairs of subtelo-acrocentric (1st to 5th), two pairs of submeta-subtelocentric (6th-7th), one submetacentric (9th) and two pairs of metacentric chromosomes (8th, 10th). A supernumerary chromosome variation (presence/absence of B-chromosome) and occurrence of triploidy in one population of I. platycephalus are reported. In addition, the karyological analysis of Lithuanian population of I. variegatus was carried out. In the light of presented and previously obtained data comparative karyology of genus Ichthyocotylurus is discussed.     

Molecular and morphological evidence suggests the reallocation from Parastrigea brasiliana (Szidat, 1928) Dubois, 1964 to Apharyngostrigea Ciurea, 1927 (Digenea: Strigeidae), a parasite of boat-billed heron (Cochlearius cochlearius) from the Neotropical region

Parastrigea brasiliana (Szidat, 1928) Dubois, 1964, was described from (Cochlearius cochlearius) in South America. The taxonomy of this species has been unstable due that it was described as a member of Strigea Abildgaard, 1790. However, the same author one year later transferred it to Apharyngostrigea Ciurea, 1927 and since then, it has been alternatively placed in the genus Apharyngostrigea or Parastrigea Szidat, 1928 from Strigeidae. In the current research, specimens identified as P. brasiliana were collected from type host in southeastern Mexico. We sequenced three molecular markers: the internal transcribed spacers ITS1 and ITS2 including the 5.8S gene (ITS region), the D1-D3 domains of the large subunit (LSU) from nuclear DNA and cytochrome c oxidase subunit I (cox 1) from mitochondrial DNA. These sequences were aligned with other sequences available in the GenBank dataset from Strigeidae. Maximum likelihood and Bayesian analyses inferred with three molecular markers consistently showed that P. brasiliana is not closely related to other members of the genus Parastrigea and are placed in a reciprocal monophyletic clade inside Apharyngostrigea, with very low genetic divergence, varying from 0 to 0.09% for the ITS, from 0 to 0.08% for the LSU and from 0.21 to 0.43% for cox 1. Consequently, we proposed to reallocate it to A. brasiliana. The phylogenetic analyses obtained are key and very useful for re-evaluate the morphology of A. brasiliana because this species share morphological characters with the genera Parastrigea (concentration of vitelline follicles distributed in two lateral expansions on the forebody) and Apharyngostrigea (absence of pharynx). Finally, the current record of A. brasiliana expands its distribution range in four countries, namely, the USA, Mexico, Venezuela and Brazil, in the Neotropical region.     

Phylogenetic relationships of Echinostoma Rudolphi, 1809 (Digenea: Echinostomatidae) and related genera re-assessed via DNA and morphological analyses

In order to investigate the relationships within the Echinostomatidae two data sets of gene sequences were analysed. The first consisted of all previously published ND1 sequences (20) together with 17 new sequences. The latter represented six species from the cosmopolitan genera Echinostoma, Echinoparyphium, Hypoderaeum and Isthmiophora. The second data-set of ITS sequences again included all previously published sequences (12) and three new sequences from species of Echinostoma, Echinoparyphium and Isthmiophora. All new isolates, as well as voucher material from five previously sequenced isolates, were identified on the basis of morphological characters. The phylogenetic trees inferred from the ND1 data set helped to clarify the generic affiliation of all isolates and confirmed the morphological identifications. The only exception was Echinoparyphium aconiatum, whose current position in the genus Echinoparyphium was not supported by the sequence data. Although the ITS data provided insufficient resolution for an unequivocal solution to the relationships within the genus Echinostoma, it supported the identification of Echinoparyphium ellisi and the distinct species status of three isolates of Echinostoma revolutum as predicted from the ND1 data.     

Histopathology, ultrastructure and immunohistochemistry of Coregonus lavaretus hearts naturally infected with Ichthyocotylurus erraticus (Trematoda)

Histopathological, ultrastructural and immunohistochemical investigations were conducted on 26 specimens of powan Coregonus lavaretus (L.) from Loch Lomond (Scotland). The hearts of all 26 powan (15 females and 11 males) investigated harboured metacercariae of the digenean trematode Ichthyocotylurus erraticus (Rudolphi, 1809). The vast majority of metacercariae were located either singly or as an aggregation of white cysts on the surface of the bulbus arteriosus. The intensity of infection ranged from 2 to 200 larvae heart(-1), although the number of metacercariae found on male powan did not exceed 13. Histochemically, the parasite cyst wall gave a strong positive reaction with periodic acid schiff (PAS) and a faint positive signal with Azan-Mallory stain. All the metacercariae cysts were embedded in a granulomatous proliferation of heart epicardium tissue, forming a reactive fibroconnective capsule around the parasite. The capsule enclosing the parasite (produced by the host's reaction to the parasite) measured 13.57 to 90.20 microm (37.43 +/- 3.56) in thickness. Within the capsule wall, eosinophilic granular cells (EGCs), granulocytes, melanocytes and, in some instances, partially degenerated or vacuolated epithelioid cells were observed in close proximity to the cyst wall. Pigment-bearing macrophages were scattered throughout the granulomatous host-tissue reaction and as macrophage aggregates (MAs) within the capsules surrounding parasites. Immunohistochemical tests were applied to infected heart sections using 12 different antisera. Nerve fibres immunoreactive to bombesin, substance P (SP), and atrial natriuretic peptide (ANP) antisera were observed in close proximity to the parasite larvae. The presence of a serotonin-like substance was also observed within host immune-cells surrounding trematode cysts. Large cells of the epicardium were found to be immunoreactive to met-enkephalin and vasoactive intestinal peptide (VIP) antisera but not immunoreactive to anti-protein gene-product 9.5 (PGP9.5) sera.     

Euclinostomum heterostomum (Rudolphi, 1809) (Trematoda): life-cycle,growth and development of the metacercaria and adult

The life-history of Euclinostomum heterostomum (Rudolphi, 1809), a clinostomid trematode occurring in the oesophagus and buccal cavity of pond heron Ardeola grayii and cattle egret Bubulcus ibis at Visakhapatnam, India, has been traced from egg to adult stage, based on field and laboratory studies. Eggs released by gravid flukes hatched into miracidia in 10 days. The snail Indoplanorbis exustus served as the first intermediate host and the intramolluscan phase, involving the development of two redial generations and cercariae, took two months. Natural infections with the metacercaria were found in the kidneys and liver of the fish Channa punctatus and muscles of C. orientalis. Laboratory experiments demonstrated that more than 2 months are required for the development of the cercaria into the infective metacercarial stage. The morphology of all of the stages in the life-cycle has been studied in detail, as well as the growth characteristics of the metacercaria within the fish host. Two phases in the metacercarial growth were noted, a lag phase followed by an exponential phase. Allometric growth parameters of body proportions and organs of the metacercaria and the adult are compared. The validity of various species included in the genus Euclinostomum is discussed.     

Description of embryonic development and ultrastructure in miracidia of Cardiocephaloides longicollis (Digenea,Strigeidae) in relation to active host finding strategy in a marine environment

The functional ultrastructure and embryonic development of miracidia in naturally released eggs of the trematode Cardiocephaloides longicollis were studied using light and transmission electron microscopy. This species has operculated eggs and embryogenesis occurs in the marine environment before an actively infecting ciliated miracidium hatches. Six different developmental stages were identified. The lack of pores in the eggshell indicates its impermeability and the miracidium's dependency on glycogen nutritive reserves, contained in numerous vitellocytes in early embryos. As the development advances, these merge into larger vitelline vacuoles that encircle the miracidium and may aid its hatching. Tissue and primary organ differentiation were observed in advanced stages, i.e., terebratorium, glands, cerebral ganglion, peripheral sensory endings, and eyespots. The anterior part of the body contains a single apical and paired lateral glands, as well as two types of sensory endings, which permit location, adhesion, and penetration of the host. No previous studies describe the embryonic development and ultrastructure of miracidia in strigeids, however, some of the structural features shared with other, well described species with unknown life cycles are emphasised. This study highlights that ultrastructural data have to be interpreted in relation to parasite biology to understand the structural requirements of specific parasite strategies.     

The immune response of the thick-lipped grey mullet, Chelon labrosus (Risso, 1826), to metacercarial infections of Cryptocotyle lingua (Creplin, 1825)

Grey mullet, Chelon labrosus , (60–100 g) have been found to respond immunologically to Cryptocotyle lingua , following a single exposure to 20000 cercariae, by the production of humoral antibody, sensitized pronephric leucocytes and cytotoxic serum factors. Antibody titres measured by passive haemagglutination reached a peak at week 4 with a -log₂ titre of 16±S.E. 1.0, and titres of 10.7±S.E. 1.0 were still recorded after 10 weeks at the termination of the experiment. Cercarial agglutination was found unreliable as a rapid test. Pronephric leucocytes, sensitive to cercarial antigen when measured by the under-agarose migration method, were detected between weeks 1 and 6, peaking at week 2. In vitro polarization was increased when cells were incubated with the antigen, but this increase was not significantly different between control and infected fish. Heat-labile cytotoxic factors of immune sera have been demonstrated to whole cercariae in vitro , these factors being associated with structural damage to the tegument. Pronephros cells isolated from immune fish during each week of infection showed no evidence of adherence to cercariae or metacercariae in vitro. The results are discussed in relation to both host response and course of infection of the parasite.     

Central European Strigeidae Railliet, 1919 (Trematoda: Strigeidida): Molecular and comparative morphological analysis suggests the reclassification of Parastrigea robusta Szidat, 1928 into Strigea Abildgaard, 1790

Strigeidae Railliet, 1919 are digenean parasites of birds and mammals that are characteristic by their cup-shaped forebody and bilobed holdfast organ. Despite that the family is taxonomically unsettled, particularly due to a very limited number of visible autapomorphic identification features, molecular phylogenetics have never been applied to analyze the relationships among European members of Strigeidae except for the genus Ichthyocotylurus. Here, we analyze the Strigeidae found during the examination of Czech birds performed from 1962 to 2017, and we provide comparative measurements and host spectra, including prevalence and intensity; we also provide and analyze sequences of four DNA loci of 12 of the Strigeidae species. We suggest the reclassification of Parastrigea robusta Szidat, 1928 as Strigea robusta (Szidat, 1928) Heneberg and Sitko, 2018 comb. n. The genera Strigea Abildgaard, 1790 and Parastrigea Szidat, 1928 appear paraphyletic, and morphological diagnostic features of genera within Strigeini Dubois, 1936 are invalid. The mute swan Cygnus olor hosts two Cotylurus spp., Cotylurus syrius Dubois, 1934 and a second species with molecular identification features shared in part with Cotylurus cornutus (Rudolphi, 1808) and Cotylurus gallinulae Lutz, 1928. New host records are provided for seven species. Analyses of non-European genera of the Strigeidae are needed to provide an updated key to Strigeini.     

Studies on the life-cycle of Ganeo tigrinus Mehra & Negi, 1928 (Digenea)

The life-history stages of Ganeo tigrinus Mehra & Negi, 1928 infecting the Indian bull frog Hoplobatrachus tigerinus (Daudin) are described, those from cercaria to egg-producing adult having been established in the laboratory. Non-virgulate xiphidiocercariae are released by the planorbid snail Indoplanorbis exustus (Deshayes). Metacercariae occur in the haemocoel of dragonfly nymphs and become infective to the frog H. tigerinus within 15 days. The pre-patent period is 45 days. Growth and development of both metacercariae and adults are described in detail. Comments on the systematic position of Ganeo Klein, 1905 are included.     

Chemical Nature and Mode of Stabilization of Egg-shell in Helicometra pulchella (Rudolphi, 1819) (Digenea: Opecoelidae)

The chemical nature and mode of stabilization of egg-shell protein in digenetic trematode Helicometra pulchella (Rudolphi, 1819) have been investigated using histochemical techniques. It was found that the egg-shell is stabilized by quinone-tanning together with dityrosine. Other structural proteins (elastin, collagen and keratin-like proteins), glycogen and acid mucopolysaccharides were absent in egg-shell. Tyrosine was present in vitelline cells and immature egg-shell indicating that the proteins involved in quinone-tanning were tyrosine rich and tyrosyl residues are modified to form dityrosine in mature egg-shell.     

New specimens and molecular data provide validation of Apatemon jamiesoni n. sp. (Trematoda: Strigeidae) from water birds in New Zealand

Systematic Parasitology - A study published in 2016 reported on an undescribed species of Apatemon (Strigeidae) from New Zealand that was previously well known from its larval stages. Only a single...