Gyrodactylus gvozdevi n. sp. (Gyrodactylidae: Monogenea) from Noemacheilus dorsalis (Kessler) in Kazakhstan

Gyrodactylus gvozdevi n. sp. (Gyrodactylidae: Monogenea) is described from the skin of the freshwater fish Noemacheilus dorsalis (Kessler) (Cobitidae: Cypriniformes) from Kazakhstan. This species is most closely related to G. pseudonemachili Ergens & Bykhovsky, 1967 in the shape and size of the anchors and both the ventral and dorsal bars, but can be distinguished from it by the shape and size of the hookproper of the marginal hooks.     

Water- and cryoprotectant-permeability of mature and immature oocytes in the medaka (Oryzias latipes)

The permeability of the plasma membrane plays a crucial role in the successful cryopreservation of oocytes/embryos. To identify a stage feasible for the cryopreservation of teleost oocytes, we investigated the permeability to water and various cryoprotectants of medaka (Oryzias latipes) oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. In sucrose solutions, the volume changes were greater in GV oocytes than MII oocytes. Estimated values for osmotically inactive volume were 0.41 for GV oocytes and 0.74 for MII oocytes. Water-permeability (microm/min/atm) at 25 degrees C was higher in GV oocytes (0.13+/-0.01) than MII oocytes (0.06+/-0.01). The permeability of MII oocytes to various cryoprotectants (glycerol, propylene glycol, ethylene glycol, and DMSO) was quite low because the oocytes remained shrunken during 2 h of exposure in the cryoprotectant solutions at 25 degrees C. When the chorion of MII oocytes was removed, the volume change was not affected, except in DMSO solution, where dechorionated oocytes shrunk and then regained their volume slowly; the P(DMSO) value was estimated to be 0.14+/-0.01x10(-3) cm/min. On the other hand, the permeability of GV oocytes to cryoprotectants were markedly high, the P(s) values (x10(-3) cm/min) for propylene glycol, ethylene glycol, and DMSO being 2.21+/-0.29, 1.36+/-0.18, and 1.19+/-0.01, respectively. However, the permeability to glycerol was too low to be estimated, because GV oocytes remained shrunken after 2 h of exposure in glycerol solution. These results suggest that, during maturation, medaka oocytes become less permeable to water and to small neutral solutes, probably by acquiring resistance to hypotonic conditions before being spawned in fresh water. Since such changes would make it difficult to cryopreserve mature oocytes, immature oocytes would be more suitable for the cryopreservation of teleosts.     

Pseudodactylogyrus kamegaii sp. n. (Monogenea: Pseudodactylogyridae) from wild Japanese eel,Anguilla japonica

The monogenean Pseudodactylogyrus kamegaii sp. n. is described, based on specimens collected from the gills of wild Japanese eel Anguilla japonica caught in Chiba Prefecture, Japan. This species is the most similar to P. anguillae (Yin and Sproston, 1948), but different in the shape and measurements of the male copulatory organ, vagina and marginal hook. This new species was collected from the eel in brackish waters, while P. anguillae and P. bini, the other known pseudodactylogyrids of Japanese eel, have been recorded only in fresh waters.     

Effects of cadmium on the reproductive axis of Japanese medaka (Oryzias latipes)

Cadmium (Cd) is a ubquitous element and a significant inorganic pollutant that has previously been found to bioaccumulate in reproductive organs of fish and disrupt important endocrine processes, especially those involved in synthesis, release and metabolism of hormones. Clearly, there is potential for reproductive effects in fish populations exposed to Cd, however, few studies have investigated the non-lethal consequences of Cd in fish. To this extent, adult male and female Japanese medaka were exposed to 0-10 ppb Cd for 7 weeks. Reproductive endpoints were monitored during weeks 6 and 7 of exposure and compared to physiological responses along the hypothalamus-pituitary-gonadal (HPG) axis, including plasma vitellogenin (VTG), hepatic estrogen receptor (ER), plasma steroids, gonadal-somatic indices (GSI), and gonadal steroid release. There were no observed effects on VTG and ER by long-term Cd exposure. However, gonadal steroid release was significantly decreased in males and females at all exposure concentrations and female plasma estradiol levels were significantly altered at concentrations higher than 5 ppb Cd. Overall, responses along the HPG axis were more sensitive to Cd exposure than the reproductive and developmental endpoints, which were not affected in this study, indicating that higher level impairment in fish might be relatively protected.     

Molecular phylogeny of the medaka fishes genus Oryzias (Beloniformes: Adrianichthyidae) based on nuclear and mitochondrial DNA sequences

The phylogenetic relationships among medaka fishes of 2 genera, Oryzias and Xenopoecilus, were studied using the nuclear tyrosinase and mitochondrial 12S and 16S rRNA genes. Of the 23 species currently described for these genera, 13 species of Oryzias and 2 species of Xenopoecilus were examined. The tree topologies obtained from the nuclear and mitochondrial data were consistent, indicating that Xenopoecilus is a polyphyletic genus nested within Oryzias. This result suggested the necessity for a systematic study and taxonomic revision of Xenopoecilus. The combined data analysis of all data partitions resulted in a well-resolved tree, with most internal branches supported by high statistical values. Based on our combined data phylogeny, we divided the Oryzias species into three major species groups, namely the latipes, javanicus, and celebensis groups. These three groups corresponded to the three chromosomal groups (biarmed, monoarmed, and fused chromosome groups) previously proposed from karyological analyses. The phylogeographic pattern suggests historical vicariance between Sulawesi Island and the continental shelf.     

Gyrodactylus longipes n. sp. (Monogenea: Gyrodactylidae) from farmed gilthead seabream (Sparus aurata L.) from the Mediterranean

Gyrodactylus longipes n. sp. (Monogenea, Gyrodactylidae) is described from the gills of farmed juvenile gilthead seabream (Sparus aurata L.) from two sites located in Italy and Bosnia-Herzegovina and represents the second species of Gyrodactylus to be described from S. aurata. Gyrodactylus orecchiae Paladini, Cable, Fioravanti, Faria, Di Cave et Shinn, 2009 was the first gyrodactylid to be described from S. aurata, from populations cultured in Albania and Croatia. In the current study, G. longipes was found in a mixed infection with G. orecchiae on fish maintained in Latina Province, Italy, thus extending the reported distribution of the latter throughout the Mediterranean. The morphology of the opisthaptoral hard parts of G. longipes is compared to those of G. orecchiae, using light and scanning electron microscopy. Gyrodactylus longipes is characterised by having larger, elongated ventral bar processes and long, triangular-shaped toe region to their marginal hook sickles which, by comparison, are rhomboid in G. orecchiae. The marginal hook sickles of G. longipes are almost double the size of G. orecchiae which allows for their rapid discrimination from each other in mixed infections. A comparison of the DNA sequence of the ribosomal internal transcribed spacer 1 and 2 regions (ITS1 and ITS2) of G. longipes with the corresponding sequence from G. orecchiae and with those available in GenBank, supports the separate species status of G. longipes. Part of this study necessitated an overview of the existing Gyrodactylus fauna from Italy and Bosnia-Herzegovina; a summary from each country is provided here to assist future investigations.     

Gyrodactylus gemini n. sp. (Monogenea: Gyrodactylidae), a parasite of Semaprochilodus taeniurus (Steindachner) from the Venezuelan Amazon

Gyrodactylus gemini n. sp. (Monogenea, Gyrodactylidae) is described from the surface of the body and fins of the fish Semaprochilodus taeniurus (Steindachner) imported into Britain from the Venezuelan Amazon. The new species differs from other species of the genus, including those described from South and Central America, by having: (i) stout hamuli with straight shafts and diverging roots; (ii) marginal hooks with the sickle length larger than the width; (iii) a dorsal bar without a medial constriction; (iv) a rectangular ventral bar with short processes; (v) a triangular ventral bar membrane; and, most obviously, (vi) at least two generations which can develop two embryos simultaneously. This is the first known species of the genus Gyrodactylus from the Venezuelan Amazon and the first record of the subgenus Gyrodactylus (Gyrodactylus) from South America.     

Influence of parental and developmental cadmium exposure on endocrine and reproductive function in Japanese medaka (Oryzias latipes)

Cadmium (Cd) is a ubiquitous element and an important anthropogenic metal contaminant. A series of assays were modified or developed for Japanese medaka (Oryzias latipes), and used to compare the effects of Cd exposure on indicators of endocrine function in adult animals previously exposed in ovo or as hatchlings. Adults were raised either from eggs produced during a 2 week exposure to 0-10 microg/l Cd or from fry exposed for 2 weeks beginning 2 days after hatching. The reproductive capacity of the resulting adults was determined during a 2 week period during which half of the animals were re-exposed to Cd. Two week Cd exposure did not result in reproductive impairment despite producing some changes in circulating steroid concentration. In addition, 1 microg/l cadmium exposure in ovo elevated male hepatic vitellogenin (VTG) relative to controls. Hence, steroid parameters were a better biomarker of cadmium exposure than changes in VTG. However, reproductive impairment was not correlated to change in VTG or plasma steroids.     

A new monogenean species,Gyrodactylus ajime n. sp. (Gyrodactylidae), parasitic on Niwaella delicata (Niwa), an endemic loach of Japan

A new gyrodactylid species, Gyrodactylus ajime n. sp., is described from the skin and fins of an endemic and commercially important loach Niwaella delicata (Niwa) (Cypriniformes: Cobitoidea: Cobitidae), from the upper reach of the River Yura in Ashiu, Kyoto Prefecture, Japan with scanning electron microscopy observations and molecular data. This species can be distinguished from congenerics by characteristics of the sickle of the marginal hook: the sickle proper is composed of two straight sections and a curved section; its short point extends beyond the limit of the toe and is directed downwards; and the upper corner of heel angular is slightly raised. There are 31 species and subspecies of native loaches (Cobitoidea) known from Japan, of which 25 are red-listed nationally, and two are commercially important. I reviewed all reports of monogeneans from Japanese loaches, and found that only five fish species have been recorded as hosts of these parasites. This suggests that the parasite fauna of these fishes is poorly described. It is urgent to thoroughly describe the monogenean parasites of loaches in Japan as some of the parasite species may be threatened with co-extinction because their hosts are threatened, and to understand any negative impacts on commercially important fish species.

     

Transient expression of foreign DNA during embryonic and larval development of the medaka fish (Oryzias latipes)

Summary Species of small fish are becoming useful tools for studies on vertebrate development. We have investigated the developing embryo of the Japanese medaka for its application as a transient expression system for the in vivo analysis of gene regulation and function. The temporal and spatial expression patterns of bacterial chloramphenicol acetyltransferase and galactosidase reporter genes injected in supercoiled plasmid form into the cytoplasm of one cell of the two-cell stage embryo was promoter-specific. The transient expression was found to be mosaic within the tissue and organs reflecting the unequal distribution of extrachromosomal foreign DNA and the intensive cell mixing movements that occur in fish embryogenesis. The expression data are consistent with data on DNA fate. Foreign DNA persisted during embryogenesis and was still detectable in some 3- and 9-month-old adult fish; it was found in high molecular weight form as well as in circular plasmid conformations. The DNA was replicated during early and late embryogenesis. Our data indicate that the developing medaka embryo is a powerful in vivo assay system for studies of gene regulation and function.This work contains part of the PhD thesis of C. Winkler     

Expression of aquaporin-3 improves the permeability to water and cryoprotectants of immature oocytes in the medaka (Oryzias latipes)

The permeability of the plasma membrane plays a crucial role in the successful cryopreservation of oocytes and embryos. Several efforts have been made to facilitate the movement of water and cryoprotectants across the plasma membrane of fish oocytes/embryos because of their large size. Aquaporin-3 is a water/solute channel that can also transport various cryoprotectants. In this study, we tried to improve the permeability of immature medaka (Oryzias latipes) oocytes to water and cryoprotectants by artificially expressing aquaporin-3. The oocytes were injected with aquaporin-3 cRNA and cultured for 6-7 h. Then, hydraulic conductivity (L(P)) and cryoprotectant permeability (P(S)) were determined from volume changes in a hypertonic sucrose solution and various cryoprotectant solutions, respectively, at 25 degrees C. The L(P) value of the cRNA-injected oocytes was 0.22+/-0.04 microm/min/atm, nearly twice larger than that of intact or water-injected oocytes (0.14+/-0.02 and 0.14+/-0.03 microm/min/atm, respectively). P(S) values of intact oocytes for ethylene glycol, propylene glycol, and DMSO were 1.36+/-0.34, 1.97+/-0.20, and 1.17+/-0.52 x 10(-3) cm/min, respectively. The permeability to glycerol could not be calculated because oocytes remained shrunken in the glycerol solution. On the other hand, cRNA-injected oocytes had significantly higher P(S) values (glycerol, 2.20+/-1.29; ethylene glycol, 2.98+/-0.36; propylene glycol, 3.93+/-1.70; DMSO, 3.11+/-0.74 x 10(-3) cm/min) than intact oocytes. When cRNA-injected oocytes were cultured for 12-14 h, 51% matured to the metaphase II stage, and 43% of the matured oocytes were fertilized and hatched following in vitro fertilization and 14 days of culture. Thus, the permeability of medaka oocytes to water and cryoprotectants was improved by the artificial expression of aquaporin-3, and the oocytes retained the ability to develop to term.     

The nuclear rDNA region of Gyrodactylus arcuatus and G. branchicus (Monogenea: Gyrodactylidae)

The primary structure of the ribosomal DNA internal transcribed spacers (ITS-1 and ITS-2) and 5.8S rRNA gene were used to characterize and identify 2 monogenean species of Gyrodacrylus living externally on the threespine stickleback (Gasterosteus aculeatus). The ITS region was amplified by PCR from freshwater, brackish, and marine isolates of Gyrodactylus arcuatus and G. branchicus, and the ends of the coding regions were identified by comparative alignment. No intraspecific and very low interspecific variation were observed in the 5.8S rRNA gene; high inter- and low intraspecific variation were revealed in the ITS-1 and ITS-2 regions. The morphological species identification was in all cases confirmed by the molecular identification. Intraspecifically, samples from 2 locations in the North Sea could be differentiated, but the Baltic sample resembled North Sea genotypes. Our approach offers perspectives for a multimetric genetical, morphometrical, and ecological taxonomy of the genus Gyrodactylus.     

Development and application of a monoclonal antibody-based sandwich ELISA for quantification of Japanese medaka (Oryzias latipes) vitellogenin

Vitellogenin (Vtg) was purified from ascitic fluid of a 17beta-estradiol (E2)-treated female Japanese medaka by anion-exchange chromatography. The molecular mass of medaka Vtg by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), corresponding to the Vtg monomer, was 200 kDa. BALB/c mice were immunized with purified-Vtg and two hybridoma clones producing specific antibodies against medaka Vtg were selected. The specificity of these monoclonal antibodies (mAbs) was evaluated by Western blot analysis of the plasma proteins separated on SDS-PAGE, and no cross-reactivity was observed with plasma proteins from control males. A sandwich enzyme-linked immunosorbent assay (ELISA) for quantification of medaka Vtg was developed using these mAbs. The assay range was between 1 and 100 ng/ml, and the intra- and inter-assay variations determined from plasma samples were within 7.7 and 8.5%, respectively. Recovery of medaka Vtg added to plasma was 92-111%. In a plasma dilution test, plots of Vtg concentration gave a straight line. After exposure of male medaka to E2 (10 ng/l), Vtg appeared in liver and plasma on the first day and reached a maximum on the 3rd to 5th day. The sandwich ELISA could be useful for the detection of estrogenic properties, and the medaka Vtg bioassay could be a very sensitive and good tool for screening of endocrine disrupting compounds.     

Taxonomy and host-specificity of Gyrodactylus aideni n. sp. and G. pleuronecti (Monogenea: Gyrodactylidae) from Pseudopleuronectes americanus (Walbaum) in Passamaquoddy Bay, New Brunswick, Canada

Wild and cultured winter flounder Pseudopleuronectes americanus (Walbaum) from Passamaquoddy Bay were surveyed for species of Gyrodactylus Nordmann, 1832. Two species were found: G. pleuronecti Cone, 1981 and G. aideni n. sp, both members of Malmberg’s ‘groenlandicus group’. Although the hard parts in the haptor are very similar in the two species, hamuli of G. aideni are consistently shorter than those of G. pleuronecti. The two species differed by 35 base pairs in the ITS 1, 5.8 and ITS 2 region. A BLAST search identified a variety of species of Gyrodactylus from marine fishes in the Atlantic Ocean as closest matches, indicating the ‘groenlandicus group’ is part of a major marine lineage within Gyrodactylus (sensu lato) that has successfully radiated among coastal percid, pleuronectid, cottid and anarhichadid fishes. Exposure experiments suggested that winter flounder is the primary host of both species of parasites and that three other pleuronectid species in the bay may potentially serve only as occasional transport hosts.     

Gyrodactylus neili n. sp. (Monogenea: Gyrodactylidae), a parasite of chain pickerel Esox niger Lesueur (Esocidae) from freshwaters of New Brunswick, Canada

Gyrodactylus neili n. sp. (Monogenea: Gyrodactylidae) is described from the fins and body surface of Esox niger Lesueur (chain pickerel) (Esocidae) from the St. Croix River drainage, New Brunswick, Canada. G.␣neili n. sp. resembles most closely G. fryi Cone & Dechtiar, 1984, a parasite of E. masquinongy in North America, in having relatively large thin hamuli, well-developed marginal hook sickles with a relatively long, wide blade and short handle, a ventral bar with small antero-lateral processes and tongue-shaped membrane, and a cirrus with many small spines in two rows. The new species is easily separated from G. fryi by the length of the hamuli (70-76 m versus 92 m, respectively), by the distal width of the sickle (7–9 versus 14-16 m, respectively) and by subtle differences in the shape of the toe and heel of the marginal hook sickle. Sequence data (922 bp) of rDNA (internal transcribed spacers 1 and 2 and 5.8S) of G. neili n. sp. returned no identical matches in GenBank. The 5.8 sequence alone, however, was identical to morphologically similar gyrodactylids of the subgenus Gyrodactylus from cyprinid fishes in Eurasia. The discovery of G. neili n. sp. and features of its genetic makeup support the idea that this lineage parasitised ancestral cyprinids and that it radiated, possibly through predator/prey interactions, to an ancestor of contemporary Esox. It is concluded that DNA comparison of monogeneans on Holarctic freshwater hosts, such as E.␣lucius, may shed light on the nature of speciation of these parasites.