Parasites in a man-made landscape: contrasting patterns of trematode flow in a fishpond area in Central Europe.

We have explored a large body of novel data focusing on small-scale temporal and spatial patterns in the composition and structure of larval trematode communities in Lymnaea stagnalis (L.) from a typical Central European agricultural landscape. The 5 eutrophic fishponds studied provide excellent environments for the development of species-rich and abundant trematode communities. Nine prevalent species were consistently present in component communities, but had differential contribution to the parasite flow in the 5 ponds resulting in significant contrasting patterns of community similarity and the prevalence of the 3 major transmission guilds driving this similarity. Component communities split into 2 groups: (i) those from the large pond dominated by anatid and larid generalists with active miracidial transmission; and (ii) those from the smaller ponds dominated by 2 plagiorchioideans infecting snails via egg ingestion. We put forward 3 hypotheses for the remarkable differences in larval trematode flow in the similar and closely located eutrophic ponds: (i) species-specific differences in parasite colonization potential displayed by an 'active-passive' dichotomy in miracidial transmission strategies of the species; (ii) top-down effects of pond context on transmission pathways of the trematodes; and (iii) competition as an important mechanism in eutrophic environments with a bottom-up effect on component community structure.

Discrimination of fish populations using parasites: Random Forests on a 'predictable' host-parasite system.

We address the effect of spatial scale and temporal variation on model generality when forming predictive models for fish assignment using a new data mining approach, Random Forests (RF), to variable biological markers (parasite community data). Models were implemented for a fish host-parasite system sampled along the Mediterranean and Atlantic coasts of Spain and were validated using independent datasets. We considered 2 basic classification problems in evaluating the importance of variations in parasite infracommunities for assignment of individual fish to their populations of origin: multiclass (2-5 population models, using 2 seasonal replicates from each of the populations) and 2-class task (using 4 seasonal replicates from 1 Atlantic and 1 Mediterranean population each). The main results are that (i) RF are well suited for multiclass population assignment using parasite communities in non-migratory fish; (ii) RF provide an efficient means for model cross-validation on the baseline data and this allows sample size limitations in parasite tag studies to be tackled effectively; (iii) the performance of RF is dependent on the complexity and spatial extent/configuration of the problem; and (iv) the development of predictive models is strongly influenced by seasonal change and this stresses the importance of both temporal replication and model validation in parasite tagging studies.

Molecules and morphology reveal cryptic variation among digeneans infecting sympatric mullets in the Mediterranean.

We applied a combined molecular and morphological approach to resolve the taxonomic status of Saccocoelium spp. parasitizing sympatric mullets (Mugilidae) in the Mediterranean. Eight morphotypes of Saccocoelium were distinguished by means of multivariate statistical analyses: 2 of Saccocoelium obesum ex Liza spp.; 4 of S. tensum ex Liza spp.; and 2 (S. cephali and Saccocoelium sp.) ex Mugil cephalus. Sequences of the 28S and ITS2 rRNA gene regions were obtained for a total of 21 isolates of these morphotypes. Combining sequence data analysis with a detailed morphological and multivariate morphometric study of the specimens allowed the demonstration of cryptic diversity thus rejecting the hypothesis of a single species of Saccocoelium infecting sympatric mullets in the Mediterranean. Comparative sequence analysis revealed 4 unique genotypes, thus corroborating the distinct species status of Saccocoelium obesum, S. tensum and S. cephali and a new cryptic species ex Liza aurata and L. saliens recognized by its consistent morphological differentiation and genetic divergence. However, in spite of their sharp morphological difference the 2 morphotypes from M. cephalus showed no molecular differentiation and 4 morphotypes of S. tensum were genetically identical. This wide intraspecific morphological variation within S. tensum and S. cephali suggests that delimiting species of Saccocoelium using solely morphological criteria will be misleading.

Follow-up trends of parasite community alteration in a marine fish after the Prestige oil-spill: shifting baselines?

This study evaluates the follow-up trends in the composition and structure of the parasite communities in the marine sparid Boops boops after the Prestige oil-spill. A total of 400 fish comprising 11 seasonal samples was analyzed from three impacted localities on the Atlantic coast of Spain. A large number of parasite species was recovered only after the spill thus suggesting a substantial alteration of the marine food webs. Post-spill communities exhibited higher richness and abundance due to the significant changes in the abundance of the common species, the latter indicating accelerated parasite transmission rates. Multivariate analyses at two nested scales detected a directional trend in parasite community succession towards the pre-spill situation, however, with no full support for community recovery. The state of parasite communities in 2005-2006 may provide the new baseline data which can serve as a framework for quantifying the impact of potential future spills in the region.

Halfway up the trophic chain: development of parasite communities in the sparid fish Boops boops.

We examined the patterns of composition and structure of parasite communities in the Mediterranean sparid fish Boops boops along a gradient of fish sizes, using a large sample from a single population. We tested the hypothesis that species forming the core of the bogue parasite fauna (i.e. species which have a wide geographical range and are responsible for recognizable community structure) appear early in the fish ontogeny. The sequential community development observed supported the prediction that core species appear in the fish population earlier than rare and stochastic species. There was also a strong correlation between the order of 'arrival' of the species and their overall prevalence. Six key species were responsible for recognizable community structure across size/age cohorts; the addition to this baseline community of key parasite species resulted in a nested structure that is linked to differential species abundance rather than fish size. Information on the life-cycles, distribution and host range of the parasites is used to explain the observed patterns of parasite community structure. We conclude that the small mouth size of B. boops coupled with suction feeding may provide a setting for passive sampling as a mechanism leading to non-random parasite community structure.

Parasite communities in Boops boops (L.) (Sparidae) after the Prestige oil-spill: detectable alterations.

Environmental pollution affects parasite populations and communities, both directly and through effects on intermediate and final hosts. In this work, we present a comparative study on the structure and composition of metazoan parasite communities in the bogue, Boops boops, from two localities (Galician coast, Spain) affected by the Prestige oil-spill (POS). We focus on the distribution of both individual parasite species and larger functional groupings by using both univariate and multivariate analyses. Our results indicate directional trends in community composition that might be related to the Prestige oil-spill disturbance of the natural coastal communities off Galicia. Endoparasite communities in B. boops reflected a notable change in the composition and abundance of the benthic fauna in the localities studied post-spill probably due to organic enrichment after the POS.

Saturnius minutus n. sp. and S. dimitrovi n. sp. (Digenea: Hemiuridae) from Mugil cephalus L. (Teleostei: Mugilidae), with a multivariate morphological analysis of the Mediterranean species of Saturnius Manter, 1969.

Three species of the bunocotyline genus Saturnius Manter, 1969 are described from the stomach lining of mugilid fishes of the Mediterranean and Black Seas. Two of the species are new: S. minutus n. sp. occurs in Mugil cephalus off the Mediterranean coast of Spain; and S. dimitrovi n. sp., a parasite of M. cephalus off the Bulgarian Black Sea coast and the Spanish Mediterranean coast, was originally described as S. papernai by Dimitrov et al. (1998). In addition, S. papernai Overstreet, 1977 is redescribed from M. cephalus off the Spanish Mediterranean coast and from Liza aurata and L. saliens off the Bulgarian Black Sea coast. The three species are distinguished morphometrically using univariate and multivariate analyses. These results were verified using Linear Discriminant Analysis which correctly allocated all specimens to their species designations based on morphology (i.e. 100% successful classification rate) and assigned almost all specimens to the correct population (locality). The following variables were selected for optimal separation between samples: the length of the forebody, ventral sucker and posterior testis, the length and width of the posteriormost pseudosegment, and the width of the muscular flange at ventral sucker level.

Robinia aurata n. g., n. sp. (Digenea: Hemiuridae) from the mugilid Liza aurata with a molecular confirmation of its position within the Hemiuroidea.

Robinia aurata n. g., n. sp. is described from Liza aurata (Mugilidae), the golden grey mullet, from the Ebro Delta, Spanish Mediterranean. The new genus differs from all other hemiurid genera in the combined possession of muscular flanges and a vestigial ecsoma. Within the Bunocotylinae, which currently accommodates 2 genera, Bunocotyle and Saturnius, the new genus exhibits a unique combination of blind caeca, Juel's organ, post-ovarian bulk of the uterus in the hind-body, and tegumental papillae surrounding the oral and ventral sucker apertures. Furthermore, Robinia n. g. differs from both Bunocotyle and Saturnius in the nature of the muscular extensions around the oral sucker, with the shape of a muscular belt in the latter and numerous muscular papillae in the former. The phylogenetic hypothesis for the Bunocotylinae developed from sequence data analyses based on partial lsrDNA and complete ssrDNA combined (22 species) and V4 domain of the ssrRNA gene (37 species) supports the erection of the new genus and confirms its position within the Hemiuroidea. Both molecular analyses confirmed the monophyly of the Hemiuroidea, its division into 2 major clades and the polyphyly of the Derogenidae, as in previous studies, and suggest that the Gonocercinae (with 2 genera, Gonocerca and Hemipera), may require a distinct familial status. Finally, there was poor support for the distinct status of the Lecithasteridae and Hemiuridae, following previous suggestions based on different sequence data sets. A key to genera of the Bunocotylinae is presented.

Microphallids in Gammarus insensibilis Stock, 1966 from a Black Sea lagoon: manipulation hypothesis going East?

Patterns of parasite site selection, variation in infection parameters and interspecific associations are examined in the light of new field data on larval microphallids in Gammarus insensibilis from a Black Sea lagoon. These patterns are discussed in relation to the predictions for the manipulative effect of Microphallus papillorobustus and its relationships with the other microphallid species based on studies on the French Mediterranean coast. Four species were recovered: Maritrema subdolum, Microphallus hoffmanni, M. papillorobustus and Levinseniella propinqua. The latter two were located in both corporal and cephalic segments, but the selection of brain appeared stronger for L. propinqua. M. subdolum was the first colonizer of amphipod population recruits, and unequivocally the dominant species in the lagoon. There was a significant positive relationship between the parasite load of all 4 species. Concurrent infections were exceedingly frequent, and no departures from random association were detected. We found no evidence that the cerebral metacercariae of M. papillorobustus consistently predict the parasite load of any of the other species in the system and identify sources for heterogeneity that may account for the differences between the Black Sea and the Mediterranean system: habitat heterogeneity, bird diversity and host-parasite systems used to infer relationships between microphallids.

Microphallids in Gammarus insensibilis Stock, 1966 from a Black Sea lagoon: host response to infection.

We examined the patterns of parasite melanization in Gammarus insensibilis using data on microphallids from Pomorie Lagoon (Black Sea) in the light of 3 predictions associated with host survival: (i) hosts invest more in defence in an environment where the likelihood for infection is higher; (ii) multiple immune challenges exhaust host reserves and result in decreased melanization rates in older hosts; (iii) host immune response is directed against the cerebral metacercariae of Microphallus papillorobustus that alter amphipod behaviour and are most detrimental to the host. G. insensibilis was capable of melanizing the metacercariae of all four species of trematodes found to be hosted by the amphipods. The frequency of melanization and mean abundance of melanized metacercariae were substantially higher than those observed in the same amphipod-gammarid system on the French Mediterranean coast. However, the rate of melanization was low and showed a significant decrease with amphipod size. Although the 4 species were differentially melanized, the host response was largely directed against Microphallus hoffmanni and M. subdolum. We suggest that (i) the lower melanization efficiency with age is due to the mode of infection, probably leading to loss of haemolymph and monopolization of the defence resources for wound healing and (ii) in the French system, host response focuses on the most prevalent and abundant species.

Morphological and molecular characterization of tetraphyllidean merocercoids (Platyhelminthes: Cestoda) of striped dolphins (Stenella coeruleoalba) from the Western Mediterranean.

Two types of tetraphyllidean merocercoids, Phyllobothrium delphini and Monorygma grimaldii, are well known from most cetaceans world-wide. The role of cetaceans in the life-cycle of these merocercoids is unclear because their specific identity is as yet unknown. The problem is compounded by poor descriptions of both merocercoids. We used light and scanning electron microscopy, and histological techniques to provide a thorough description of merocercoids collected from 11 striped dolphins, Stenella coeruleoalba, from the Spanish Mediterranean. We also described, for the first time, specimens of P. delphini with immature proglottides. Our merocercoids were morphologically similar to those described previously, except in the structure of the apical organ. Intra- and inter-sample variability in the morphology of the apical organ suggested that it degenerates during larval development. A subsample of 16 specimens of P. delphini and M. grimaldii was characterized for the D2 variable region of the large subunit ribosomal RNA gene (LSU) and compared with published tetraphyllidean cestode LSU sequences. P. delphini showed 2 unique signatures that differed from one another by a single base, whereas all sequences of M. grimaldii were identical. This suggests that each type may represent a single species, contrary to previous speculations based on morphological data. All merocercoid specimens formed a clade together with Clistobothrium montaukensis. Based on the low degree of divergence, all specimens of this clade are predicted to be congeneric.

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.

Helminths of the sand lizard, Lacerta agilis (Reptilia, Lacertidae), in the Palaearctic: faunal diversity and spatial patterns of variation in the composition and structure of component communities.

We studied variation in the structure of component communities of helminths in sand lizards, Lacerta agilis, from 30 localities in the Ukraine and Bulgaria. Thirty-five separate samples of lizards, with a total of 661 completely censused infracommunities, yielded 30 helminth species (4 cestodes, 10 trematodes, 3 acanthocephalans and 13 nematodes). In its range within the Ukraine, L. agilis serves as the final host for 13 species of which only 3 (S. lacertae, S. hoffmanni and P. molini) can be considered as lizard specialists. A characteristic feature of these helminth component communities was the large proportion of heteroxeneous helminth species for which L. agilis serves as paratenic host. Sand lizards in the meadow steppeland zone were primarily parasitized by larval helminths that represented a major proportion of the total number of all worms recovered while those sampled in the grassland/forest transition zone were characterized by substantially higher proportions of adult helminths using lizards as final hosts. However, L. agilis was parasitized by a much higher proportion of lizard specialists in the 'typical' habitats of the meadow steppeland zone as opposed to those located in 'marginal' habitats in the grassland/forest transition zone, where helminths were shared to a greater extent with amphibian hosts.

A redescription of Uroproctepisthmium bursicola (Creplin, 1837) n. comb. (Digenea: Echinostomatidae), and re-evaluations of the genera Episthmium Lühe, 1909 and Uroproctepisthmium Fischthal & Kuntz, 1976.

Re-examination of the type and newly collected material of Episthmium bursicola (Creplin, 1837) revealed the presence of a uroproct. The species is redescribed and transferred to the genus Uroproctepisthmium as U. bursicola n. comb. E. proximum Travassos, 1922 is also transferred to Uroproctepisthmium as U. proximum n. comb. The generic diagnosis of Uroproctepisthmium is redefined and Episthmium is tentatively retained as a synonym of Echinochasmus, following Odhner (1910), until a thorough revision of its constituent species is made.

Psilolintonum lineatum (Linton, 1928) (Echinostomatoidea: Psilostomidae) re-allocated to Podocotyle (Allocreadioidea: Opecoelidae) on the basis of a re-examination of the type material.

The type-material of Psilostomum lineatum Linton, 1928 was re-examined and identified as Podocotyle reflexa (Creplin, 1825). This re-allocation of the type and only speciesinvalidates the genus Psilolintonum that is now considered a synonym of Podocotyle.

Redescriptions of two echinostomes from freshwater fishes, with comments on Singhia Yamaguti, 1958 and Caballerotrema Prudhoe, 1960 (Digenea: Echinostomatidae).

The type-species of Singhia and Caballerotrema (Digenea: Echinostomatidae), parasites of freshwater fishes which have previously been assigned to the subfamily Singhiinae Yamaguti, 1958, are redescribed. One of the specimens of the type-series of Caballerotrema brasiliense Prudhoe, 1960 was identified as C. aruanense Thatcher, 1980. C. arapaimense Thatcher, 1980 is considered a species inquirenda. Himasthla piscicola Stunkard, 1960 is transferred to Caballerotrema as C. piscicola n. comb. The generic diagnoses of Singhia and Caballerotrema are redefined and Singhia is re-allocated to the Echinostomatinae Looss, 1899, the Singhiinae becoming a synonym of the latter.

Re-validation of Echinostoma miyagawai Ishii, 1932 (Digenea: Echinostomatidae) on the basis of the experimental completion of its life-cycle.

The life-cycle of Echinostoma miyagawai, a Eurasian species closely related to E. revolutum, was completed in the laboratory, and the morphology of the larval stages and the adults obtained experimentally was studied. Planorbis planorbis and Anisus vortex were the first intermediate hosts in the brackish Lake Durankulak on the Bulgarian Black Sea coast. Characteristic features of the cercaria include: a prominent collar with 37 spines; a tail as long as the body and with seven conspicuous fin-folds, the two ventral fin-folds being very close to each other; and a specific number and distribution of both the para-oesophageal gland-cell outlets and sensilla. The adult is characterised by: a very elongate body with a constriction at the posterior border of the ventral sucker; a large head collar with relatively small spines; a spherical ventral sucker which is only about half the maximum body width; a long cirrus-sac reaching posteriorly dorsal to the middle of the ventral sucker; indented subglobular testes; and a vitellarium forming two lateral fields of follicles which are almost confluent in the post-testicular space. The species described in this study resembles E. miyagawai, as described by Kosupko, in the morphology of larval stages and both the site and the general morphology of the adults. It differs from both E. revolutum, as described by both Kanev and Nasincová, and E. echinatum (also referred to as E. lindoense and E. barbosai by Kanev). The re-examination of Kanev's voucher specimens from his experimental studies used in his delimitation of E. revolutum and E. echinatum showed that the specimens identified by him as E. revolutum represent two distinct forms which consistently differ both from each other and from the redescription of E. revolutum which was based upon them. It also revealed that a number of specimens were wrongly identified and erroneously treated as E. echinatum by Kanev and co-workers; these include members of different genera (Hypoderaeum and Echinoparyphium) and an Echinostoma species of the group possessing 47 collar spines. The relative merits of the features used by Kanev and co-workers in discriminating the closely related Echinostoma spp. are discussed in detail with respect to the experimental evidence provided by these authors.

A quantitative approach to the evaluation of the morphological variability of two echinostomes, Echinostoma miyagawai Ishii, 1932 and E. revolutum (Frölich, 1802), from Europe.

A comparative morphometric analysis was conducted on two European species of Echinostoma in order to examine the degree of the variability in the metrical characteristics of the adults and to assess their value in discriminating species. Adult E. miyagawai and E. revolutum, obtained experimentally, were compared using univariate and multivariate statistical analyses of 35 and 25 metrical characters, respectively. All subsets of worms of different ages represented homogeneous samples with respect to their morphometric characteristics; however, univariate analyses revealed significant differences in 22 and 23 variables between the corresponding age subsets of the two species, and it was found that the different allometric growth patterns contribute to this. The variables, body width at the posterior border of the ventral sucker, pharynx length and width, ovary length, testes length and width and length of the pre-ovarian region, exhibited isometric or positive allometric growth in E. miyagawai and negative allometry in E. revolutum. A cluster analysis based on 61 specimens and 25 variables separated E. revolutum and E. miyagawai unambiguously, producing an exact ordering of the specimens with respect to their identity and age. A forward stepwise discriminant analysis identified five variables (body width at the posterior border of ventral sucker, head collar width, length of oesophagus, width of ventral sucker and length of the pre-ovarian region) which yielded a 100% accurate classification of the two species. We suggest, therefore, that the morphometric characteristics of the adult worms should be used in studies attempting the identification of species or isolates of Echinostoma spp. More comparative data need to be gathered in order that the species boundaries within the 'revolutum' group be defined more accurately.

Altered vitronectin receptor (alphav integrin) function in fibroblasts adhering on hydrophobic glass.

Function of integrins is crucial for adhesion, movement, proliferation, and survival of cells. In a recent study we found impaired fibronectin receptor function on hydrophobic substrata (G. Altankov et al. J Biomater Sci Polym Edn 1997;8:712-740). Here, we have studied the distribution and function of the vitronectin receptor (alphav integrin) in fibroblasts adhering on hydrophilic glass and hydrophobic octadecyl glass (ODS). The morphology of fibroblasts and the organization of actin cytoskeleton were studied and found to be altered on ODS, where the cells did not spread and possessed condensed actin. Pretreatment of the surfaces with serum or pure vitronectin improved cell morphology on both substrata, resulting in the development of longitudinal actin stress fibers. It was found with biotinylated vitronectin that comparable quantities of vitronectin were adsorbed from single vitronectin solutions or serum on glass and on hydrophobic ODS. The organization of the vitronectin receptors on the ventral cell surface was investigated in permeabilized cells showing normal focal adhesions in fibroblasts plated on glass but none of these structures on ODS. The distribution of alphav integrin on the dorsal cell surface was studied on nonpermeabilized living cells after antibody tagging. While fibroblasts adhering on plain or serum-treated glass developed a linear organization of alphav integrin, cells on plain and serum-treated ODS were not able to reorganize the vitronectin receptor. Studies on signal transduction with antiphosphotyrosine antibodies revealed co-localization of alphav integrin and phosphotyrosine in focal adhesions on glass and serum-treated glass. However, signaling was almost absent on plain ODS and weak on serum-treated ODS. It was concluded that alterations in vitronectin receptor function on the ventral cell surface caused by the hydrophobic material surface inhibit signal transfer and subsequent intracellular events that are important for the organization and function of integrins.

Preparation of PEG-coated surfaces and a study for their interaction with living cells.

Cell-biomaterial interaction is of great importance for the development of bioinert as well as of hybrid surfaces. This study represents our results of human fibroblast interaction with PEG-coated surfaces of differing length and structure (linear or branched) of the oxyethylene chain. We employed three PEGs -- PEG 1500 and PEG 6000, both lineal but with different chain lengths, and PEG 12500 which was branched. The PEGs were deposited on silica plates using branched poly(ethylene imine) as an anchoring polymer. Fibroblasts were plated and studied by immunofluorescence to evaluate the overall cell morphology, the organisation of the actin cytoskeleton, and the beta1-integrin (fibronectin receptor). The particular effect of fibronectin (FN) pre-adsorption was studied. Our results suggest that PEG 6000 surface is to be preferable with respect to the initial interaction with the cells. The overall cell morphology was almost normal on bare surfaces. FN pre-coating additionally improved cell adhesion and spreading as well as the organization of the actin cytoskeleton and focal adhesion formation; the PEG 12500 surface showed relatively poor initial properties. Almost no cell spreading was found on the bare surface, but FN pre-adsorption completely restored normal cell morphology. In contrast, PEG 1500 had to be considered is 'the worst' material, because of lower initial cell adhesion and spreading and FN pre-adsorption did not restore normal cell morphology.