Description of Gyrodactylus banmae n. sp. (Monogenea,Gyrodactylidae) parasitic on zebrafish,Danio rerio

A new species of Gyrodactylus was described on the body surface of zebrafish (Danio rerio) in China. Basing on morphological characteristics and ITS sequence, we identified the parasite as a new member of the Gyrodactylus-wageneri group. Morphologically, Gyrodactylus sp. nov. is greatly similar to “G. zebrae”, another species parasitic on zebrafish: both have moderately stout hamulus, marginal hook sickle with a prominent heel and toe, as well as a curved blade. However, distinct haptoral shape differences were detected between these two species. The dorsal bar is straight in Gyrodactylus sp. nov. but strongly curved in “G. zebrae”, and the sickle shaft in Gyrodactylus sp. nov. is approximately perpendicular to the base, but in “G. zebrae” it is slanted downwards. The ITS1-5.8S-ITS2 sequence also indicate that Gyrodactylus sp. nov. exhibits the highest similarity to “G. zebrae”: 95.7% sequence identity suggests interspecific differentiation. Phylogenetic analysis of the ITS1-ITS2 sequence showed that Gyrodactylus sp. nov. formed a sister clade with “G. zebrae”, and exhibited a relatively close phylogenetic relationship with G. kobayashii, G. gurleyi, and G. longoacuminatus, all of which parasitise on goldfish, Carassius auratus. To test the susceptibility of zebrafish and goldfish to the Gyrodactylus sp. nov., ten gyrodactylids were inoculated to the caudal fin of zebrafish and goldfish. The gyrodactylids exhibited the ability to attach themselves to the goldfish, and some gyrodactylids reproduced a few days after the inoculation. On day 9, however, the mean abundance sharply decreased to zero on goldfish and increased to more than 30 on zebrafish. The result suggested that golfish is an unsuitable host for Gyrodactylus sp. nov. Therefore, on the basis of morphology, molecular sequence similarity, and host susceptibility, we conclude that the gyrodactylid found on the zebrafish is a new species, which we named Gyrodactylus banmae.     

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.     

Speciation by host switch and adaptive radiation in a fish parasite genus Gyrodactylus (Monogenea,Gyrodactylidae)

Four hundred Gyrodactylus species have been formally described, but the estimated number of species in this fish ectoparasite genus of Monogenean Platyhelminthes is more than 20,000. The unusually high species richness has lead to the hypotheses of speciation and adaptive radiation via host switching. These hypotheses were tested by reconstructing a molecular phylogeny for the subgenus G. (Limnonephrotus) which is a group of freshwater parasites, including five species infecting wild and farmed salmonids. The highly variable ITS1 and ITS2 segments and the conservative 5.8S ribosomal gene were sequenced in 22 species plus two species representing the subgenus G. (Paranephrotus) as an outgroup. The phylogeny was compared with host systematics: the species were collected from six fish families (Cyprinidae, Salmonidae, Percidae, Esocidae, Gasterosteidae, and Gobitidae). The phylogenetic analysis demonstrated that G. (Limnonephrotus) is a monophyletic group that was originally hosted by cyprinids. The speciation has occurred in two episodes, the older one manifested in genetic distances 25-33% (4-6 Myr BP). The latter speciation burst occurred in one clade only, perhaps one million years ago. This clade has been morphologically identified as a wageneri species group. It is a monophyletic group of 18 species [studied here] and contains all five salmonid parasites, but also parasites, on cyprinids, percids, esocids, and gasterosteids. In G. (Limnonephrotus), eight host switches crossing the host family barrier were observed, and at least three of them were followed by repetitive speciation. Seven host-switch events were statistically confirmed by bootstrapping. The suggested model of speciation by host switch was accepted, and interestingly the adaptive radiation seems to be a consequence of host switch to a new family (key innovation model). The molecular and ecological evolution rate of Gyrodactylus parasites is manyfold in comparison to host species, and the phylogenies are largely independent and disconnected.     

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.     

Heterochronic differences in fin development between latitudinal populations of the medaka Oryzias latipes (Actinopterygii: Adrianichthyidae)

Heterochrony is believed to have played important roles in macroevolutionary morphological changes. However, few studies have focused on intraspecific heterochrony, although interspecific differences ultimately originated from variation within ancestral species. We have demonstrated heterochrony in fin development between two latitudinal populations of the medaka, Oryzias latipes . Comparisons of fin length (anal and dorsal) among wild individuals revealed that fins are shorter with respect to body length in the northern population, indicating that they are 'paedomorphic' compared with the southern population. Observations of fin ray formation and subsequent fin growth in the laboratory revealed that the timing of pterygiophore development occurs later, and that fins start to elongate later with respect to body length in the northern fish, indicating that fin growth is 'post-displaced' compared with the southern population. In addition, the rate of fin growth with respect to body length was lower in the northern males, indicating 'neoteny'. Given that all Oryzias except O. latipes are distributed in the tropics, it is likely that higher-latitude fish have evolved post-displacement and neoteny during northern extension of their geographic range. The delayed development in higher-latitude fish is probably a trade-off for faster body growth, which has evolved as an adaptation to seasonally time-constrained environments.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 571–580.     

The description of Gyrodactylus mulli sp. n. (Monogenea: Gyrodactylidae) from the Black Sea blunt-snouted mullet Mullus barbatus ponticus

A new species Gyrodactylus mulli sp. n. is described from the thorax fins of blunt-snouted mullet Mullus barbatus ponticus by the collections of B. E. Bychowsky made in 1947 near Karadag on the Black Sea. Gyrodactylus mulli sp. n. differs from G. alviga Dmitrieva et Gerasev, 2000 (described from blunt-snouted mullet too) in having another morphotype of the marginal hooks. The new species differs from G. proterorhini Ergens, 1967, which has the identical type of the marginal hooks, in having longer membrane of the ventral connective bar, longer point of the anchors, shorter marginal hooks, and lesser size of the cirrus.     

Gyrodactylus anarhichatis n. sp. and G. microanchoratus n. sp. (Monogenea: Gyrodactylidae) from the Atlantic wolf-fish Anarhichas lupus (Anarhichadidae) off the coast of Norway

The gyrodactylid monogeneans Gyrodactylus anarhichatis n. sp. and G. microanchoratus n. sp. are reported from the skin and fins of wild Atlantic wolf-fish Anarhichas lupus L. caught off the coast of northern Norway. G. anarhichatis n. sp. is also reported from farmed Atlantic wolf-fish and spotted wolf-fish A. minor Olafsen. G. anarhichatis n. sp. most closely resembles G. corti Mizelle & Kritsky, 1967, but is also similar to G. perlucidus Bychowsky & Poljansky, 1953 and G. errabundus Malmberg, 1970, while G. microanchoratus n. sp. differs from most other Gyrodactylus spp. by having marginal hooks longer than anchors and a peculiar lump on the marginal hook sickle toe pointing towards the sickle point.     

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.