Kristína Civáňová

Monogeneans of West African Cichlid Fish: Evolution and Cophylogenetic Interactions

The goals of this paper were to investigate phylogenetic and evolutionary patterns of cichlid fish from West Africa and their Cichlidogyrus and Scutogyrus monogenean parasites, to uncover the presence of host-parasite cospeciation and to assess the level of morphological adaptation in parasites. This required the following steps, each one representing specific objectives of this paper: (1) to build phylogenetic trees for Cichlidogyrus and Scutogyrus species based on ribosomal DNA sequences, (2) to investigate phylogenetic relationships within West African cichlid fish based on the analysis of mitochondrial cytochrome b DNA sequences, (3) to investigate host-parasite cophylogenetic history to gain clues on parasite speciation process, and (4) to investigate the link between the morphology of the attachment apparatus and parasite phylogeny. Phylogenetic analyses supported the monophyletic origin of the Cichlidogyrus/Scutogyrus group, and suggested that Cichlidogyrus is polyphyletic and that Scutogyrus is monophyletic. The phylogeny of Cichlidae supported the separation of mouthbrooders and substrate-brooders and is consistent with the hypothesis that the mouthbrooding behavior of Oreochromis and Sarotherodon evolved from substrate-brooding behavior. The mapping of morphological characters of the haptor onto the parasite phylogenetic tree suggests that the attachment organ has evolved from a very simple form to a more complex one. The cophylogenetic analyses indicated a significant fit between trees using distance-based tests, but no significant cospeciation signal using tree-based tests, suggesting the presence of parasite duplications and host switches on related host species. This shed some light on the diversification process of Cichlidogyrus species parasitizing West African cichlids.

Parasite diversity and microsatellite variability in native and introduced populations of four Neogobius species (Gobiidae)

Species introduced into new areas often show a reduction in parasite and genetic diversity associated to the limited number of founding individuals. In this study, we compared microsatellite and parasite diversity in both native (lower Danube) and introduced populations of 4 Ponto-Caspian gobies, including those (1) introduced from within the same river system (middle Danube; Neogobius kessleri and N. melanostomus), and (2) introduced from a different river system (River Vistula; N. fluviatilis and N. gymnotrachelus). Microsatellite data confirmed the lower Danube as a source population for gobies introduced into the middle Danube. Both native and introduced (same river system) populations of N. kessleri and N. melanostomus had comparable parasite species richness and microsatellite diversity, possibly due to multiple and/or continual migration/introduction of new individuals and the acquisition of local parasites. Reduced parasite species richness and microsatellite diversity were observed in introduced (different river system) populations in the Vistula. A low number of colonists found for N. fluviatilis and N. gymnotrachelus in the Vistula potentially resulted in reduced introduction of parasite species. Insufficient adaptation of the introduced host to local parasite fauna, together with introduction into an historically different drainage system, may also have contributed to the reduced parasite fauna.

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.

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.

Genomic Porosity between Invasive Chondrostoma nasus and Endangered Endemic Parachondrostoma toxostoma (Cyprinidae): The Evolution of MHC IIB Genes

Two cyprinid species, Parachondrostoma toxostoma, an endemic threatened species, and Chondrostoma nasus, an invasive species, live in sympatry in southern France and form two sympatric zones where the presence of intergeneric hybrids is reported. To estimate the potential threat to endemic species linked to the introduction of invasive species, we focused on the DAB genes (functional MHC IIB genes) because of their adaptive significance and role in parasite resistance. More specifically, we investigated (1) the variability of MHC IIB genes, (2) the selection pattern shaping MHC polymorphism, and (3) the extent to which trans-species evolution and intergeneric hybridization affect MHC polymorphism. In sympatric areas, the native species has more diversified MHC IIB genes when compared to the invasive species, probably resulting from the different origins and dispersal of both species. A similar level of MHC polymorphism was found at population level in both species, suggesting similar mechanisms generating MHC diversity. In contrast, a higher number of DAB-like alleles per specimen were found in invasive species. Invasive species tended to express the alleles of two DAB lineages, whilst native species tended to express the alleles of only the DAB3 lineage. Hybrids have a pattern of MHC expression intermediate between both species. Whilst positive selection acting on peptide binding sites (PBS) was demonstrated in both species, a slightly higher number of positively selected sites were identified in C. nasus, which could result from parasite-mediated selection. Bayesian clustering analysis revealed a similar pattern of structuring for the genetic variation when using microsatellites or the MHC approach. We confirmed the importance of trans-species evolution for MHC polymorphism. In addition, we demonstrated bidirectional gene flow for MHC IIB genes in sympatric areas. The positive significant correlation between MHC and microsatellites suggests that demographic factors may contribute to MHC variation on a short time scale.

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.

Patterns of parasite distribution in the hybrids of non-congeneric cyprinid fish species: is asymmetry in parasite infection the result of limited coadaptation?

Hybrids and their parasite diversity represent interesting models for evolutionary ecology. The modified immune response, shifted ecology, inheritance, and maternal ancestry of hybrid host fish are supposed to affect the diversity of their parasite communities. The pattern of metazoan parasite distribution in non-congeneric cyprinids - common bream (Abramis brama) and roach (Rutilus rutilus) (species with different morphology and ecology, and harbouring different specific parasites) - and their hybrids was analysed. Four static alternative scenarios based on parasite infection levels in hybrids and parental taxa are known. The hybrid resistance scenario predicts that hybrids are more resistant than parental taxa, resulting in low parasite infection in hybrids. This scenario is principally consistent with hybrid heterosis advantage. In accordance with this prediction, metazoan parasite abundance and prevalence were higher in parental species when compared with their hybrids. Alternatively, the dynamic Red Queen scenario of infection in hybridising systems predicts parasite adaptation to common hosts. Temporal (six sampling events) and spatial (two sampling sites) aspects as possible factors influencing parasite distribution were analysed. We found no support for this hypothesis, i.e. no changes in the frequency of hybrids or their parental species and no changes in parasite infection in parental species or hybrids were found in the different time periods. The effect of maternal ancestry on infection level was evident; hybrids exhibiting common bream mtDNA were more strongly parasitized by digeneans and crustaceans than hybrids exhibiting roach mtDNA. Hybrids harboured a majority of the specific parasites of both parental species; however, the level of infection of common bream-specific parasites (especially monogeneans) in hybrids was low. Such an asymmetrical distribution of parental species-specific parasites in hybrids may suggest the limited inheritance of protective immunological mechanisms from one parental species and reveal stronger coadaptation between common bream and its specific parasites.

Phylogenetic relationships between pinworms (Nematoda: Enterobiinae) parasitising the critically endangered orang-utan, according to the characterisation of molecular genomic and mitochondrial markers

Pinworms (Nematoda: Enterobiinae) include 52 species parasitising primates throughout the world. In the present study, we performed the first ever molecular analysis to investigate the phylogenetic position of recently described pinworms parasitising the Sumatran orang-utan. The phylogenetic analysis of mitochondrial CO1 and chromosomal 18S rDNA and ITS1 regions could support the independent status of several Nematoda species. Our molecular data clearly suggest that Enterobius (Colobenterobius) buckleyi and Lemuricola (Protenterobius) pongoi together with Pongobius hugoti form separate clades among other studied species, which significantly supports the hypothesis of recently described new species parasitising the orang-utan (Pongo abelii and Pongo pygmaeus). The phylogenetic tree based on cytochrome oxidase 1 (CO1) gene variability showed possible close relationships between L. (Protenterobius) pongoi and P. hugoti; thus, we can assume that these species could have initially diverged in sympatry from a common ancestor.

Modified sample preparation from ethanol fixed material for identification of Eudiplozoon nipponicum as a case parasite species for the combined morphological and molecular characterization

This study is focused on feasibility of two treatments of alcohol-fixed monogenean parasites that are intended to be used for the combined - molecular and morphological characterization. Conclusions of particular identification approaches highly correspond and could be used in subsequent phylogenetic studies.