An Ultrastructural Study of the Myxosporeans,Sphaerospoa angulata and Sphaerospora carassii,in the Common Carp,Cyprinus carpio L.1

Sporogenesis of Sphaerospora angulata occurs in the lumen of renal tubules, and of Sphaerospora carassii sporogenesis occurs in the gill epithelium of carp (Cyprinus carpio) from Hungarian pond farms. In infected fish, the lumen of the renal tubules is filled with disporous pansporoblasts of S. angulata. Spores are formed through the participation of paired valvogenic and capsulogenic cells, and a dikaryotic sporoplasm, all of which lie in the cytoplasm of an envelope cell. The polar filament is formed through the apparent invagination of components of the elongate external tubule into the capsular primordium, coupled with the incorporation of dense material from the lumen of the primordium and external tubule. The cytological events leading to spore formation in monosporous pansporoblasts of S. carassii are similar to those of S. angulata and most other species of Myxosporea thus far. While neither parasite appears to induce severe pathogenesis, they may contribute to morbidity in concurrent infections with other microorganisms. Since spores of S. angulata and S. carassii are not formed in plasmodia and the earliest stage observed in this study was 2-celled, earlier stages of these parasites must occur elsewhere in the carp host.     

PKX,the Causative Agent of Proliferative Kidney Disease (PKD) in Pacific Salmonid Fishes and Its Affinities with the Myxozoa1

Proliferative kidney disease (PKD), caused by an unclassified protozoan (PKX), is reported from Pacific salmon, Oncorhynchus tshawytscha (Walbaum) and O. kisutch (Walbaum), and steelhead trout, Salmo gairdneri Richardson, held at the Mad River Hatchery in California, USA. The cumulative mortality attributed to the disease was 95, 13, and 18% respectively. The mortalities were greatest at mean water temperatures of 12-14°C during July 1983. The ultrastructure of the PKX organism and its associated pathology during clinical disease in all three species were consistent with those of the parasite in rainbow trout (Salmo gairdneri) as described in European outbreaks. Significant mortalities did not occur after August, at which time the parasite could no longer be detected in the salmon species. The steelhead continued to exhibit parasites in the kidney interstitium and epithelium and lumens of the tubules. Myxosporidan trophozoites and developing spores were also observed in the lumens of the kidney tubules of these fish. Although a mixed infection with another parasite may have occurred, evidence suggests that the myxosporidans are later stages of PKX. They were only observed in fish exposed to water with the infective stage and were particularly prominent in recovering fish. The PKX organism is similar to UBO, an unclassified protozoan of carp suspected to be an early stage of Sphaerospora renicola Dyková & Lom. Both parasites infect the blood and kidney, divide by endogeny, and are released by disintegration of the primary mother cell. The intraluminal myxosporean forms show similarities to Sphaerospora spp. in that they are monosporous and sporoblasts are formed within pseudoplasmodia. It is possible that PKX migrates to the lumen of the kidney tubule and subsequently sporulates. If the myxosporean forms are later stages of PKX, then it would belong to the phlyum Myxozoa.     

Kidney sphaerosporosis with extrasporogonic stages in the blood in Baryancistrus sp. (Loricaridae) from Amazonian Brazil

Examined fish of the genus Baryancistrus (Loricaridae) from Rio Xingu in central amazonian Brazil were found infected with extrasporogonic stages of Sphaerospora sp. (Myxosporea) in the blood and sporogonic stages in the glomeruli with the resulting escape of the sporogony products into the renal tubules, apparently when the mesangial tissue in the Bowman's capsule collapsed. Spores were seen evacuating via the urinary tubules. Blood stages were initially 2.1 x 2.8 to 2.8 x 2.8 microns in size, and when fully differentiated reached 5.6 x 5.6-5.6 x 7.0 microns; in blood smears they were 11.2 x 8.4 to 14 x 8.4 microns. Bisporoblasts in the glomerular taft were 9.8 x 7.0 microns, premature spores were 5.6 x 4.2-5.6 microns.     

Light and Electron Microscope Observations On Presporogonic and Sporogonic Stages of Sphaerospora Epinepheli (Myxosporea) In Grouper (Epinephelus Malabaricus)

ABSTRACT. Presporogonic (blood) stages of Sphaerospora epinepheli Supamattaya, Fischer-Scherl. Hoffmann, Boonyaratpalin, 1990 were observed in the circulating blood, sinus of kidney, glomerurar capillaries and liver arteries of grouper Epinephelus malabaricus. the earliest detectable stage was a primary cell with one secondary cell. After cell divisions, nine to 16 secondary cells were found in one primary cell. Ultrastructural examination revealed electron-dense bodies (118-145 nm) in the cytoplasm of primary cells. Sporogonic stages and spores were located in Bowman's space and in kidney tubule lumens. Electron micrographs revealed a similar pattern of spore development as described from other Sphaerospora spp. Kidneys infected with S. epinepheli showed highly vacuolated tubular epithelial cells and severely affected renal corpuscles.     

Species of the genus Chloromyxum Mingazzini (Myxozoa: Myxosporea) infecting burbot (Lota lota L.)

Three different species of the genus Chloromyxum Mingazzini were found in burbot (Lota lota L.) collected in south-west Bohemia (Czechoslovakia). Comparison with existing records revealed that one species could be identified as C. pseudomucronatum Kashkovskiy, 1966. Found in the urinary bladder, it had subspherical spores with fine surface ridges and polysporic plasmodia. The two other myxosporea were established as new species: C. lenorae n. sp. was found in the kidney, renal corpuscles, renal tubules and interstitium, and had ellipsoid spores with surface ridges barely perceptible in the light microscope but clearly revealed by transmission electron microscopy. In the polysporic plasmodia, spores developed in pansporoblasts. C. reticulatum n. sp. was found in the gall bladder. It had polysporic plasmodia and spherical spores (average size 8.1 m in diameter) with a unique surface structures: elevated crests marking off irregular fields which appeared as a reticulum. In five of the fish infected with C. lenorae, bloodstream myxosporean stages of an extrasporogonic cycle were found. Further research is needed to determine whether they are stages of Sphaerospora cristata Shulman, 1962, a species also found in two of the burbot examined, or stages belonging to the Chloromyxum life cycle.     

Sphaerospora epinepheli N. Sp. (Myxosporea: Sphaerosporidae) Observed in Grouper (Epinephelus malabaricus)

Sphaerospora epinepheli n. sp. is described from grouper, Epinephelus malabaricus , in cage-cultured and wild fish collected from both coastal lines of southern Thailand. Subspherical to spherical spores and mono- or disporous pseudoplasmodia were observed in the lumen of kidney tubules. Pseudoplasmodia were round to elongate, size range 15.6–22.9 μm (length) × 8.4–21.6 μm (width). Spores were 7.8–10.0 μm (length) × 12.3–14.5 μm (thickness), and 7.0–9.5 μm (width) with two spherical polar capsules of equal size measuring 2.9–4.4 μm in diameter and containing polar filaments with six or seven windings. Two uninucleate sporoplasms showed iodine vacuoles. Blood stages, similar to C-blood protozoans observed from freshwater fish in Europe, were found from peripheral blood smears of grouper. Ultrastructural studies of blood stages showed a similar structure to unidentified mobile protozoans from the blood of carp. Electron dense bodies were observed in the cytoplasm of the primary cell blood stages. Infected proximal-tubular epithelial cells showed highly vacuolated cytoplasm and pycnotic nuclei.     

Sphaerospora epinepheli n. sp. (Myxosporea: Sphaerosporidae) observed in grouper (Epinephelus malabaricus)

Sphaerospora epinepheli n. sp. is described from grouper, Epinephelus malabaricus, in cage-cultured and wild fish collected from both coastal lines of southern Thailand. Subspherical to spherical spores and mono- or disporous pseudoplasmodia were observed in the lumen of kidney tubules. Pseudoplasmodia were round to elongate, size range 15.6-22.9 microns (length) x 8.4-21.6 microns (width). Spores were 7.8-10.0 microns (length) x 12.3-14.5 microns (thickness), and 7.0-9.5 microns (width) with two spherical polar capsules of equal size measuring 2.9-4.4 microns in diameter and containing polar filaments with six or seven windings. Two uninucleate sporoplasms showed iodine vacuoles. Blood stages, similar to C-blood protozoans observed from freshwater fish in Europe, were found from peripheral blood smears of grouper. Ultrastructural studies of blood stages showed a similar structure to unidentified mobile protozoans from the blood of carp. Electron dense bodies were observed in the cytoplasm of the primary cell blood stages. Infected proximal-tubular epithelial cells showed highly vacuolated cytoplasm and pycnotic nuclei.     

Chloromyxum inexpectatum n. sp. and Sphaerospora colomani n. sp. (Myxozoa: Myxosporea), parasites of the urinary system of the sterlet,Acipenser ruthenus L.

During the parasitological examination of sterlets, Acipenser ruthenus L., caught for breeding, the developmental stages of two new coelozoic species of Myxosporea were demonstrated in the urinary passages. Chloromyxum inexpectatum n. sp. occurs attached to the epithelial cells of the ureters and urinary bladder. Sphaerospora colomani n. sp. forms bisporoblastic pseudoplasmodia in the lumen of the renal tubules. A further myxosporean species, Zschokkella sturionis Tripathi, 1948, was found in the biliary ducts. The occurrence of this parasite in sterlet has not been reported so far. Examination of blood smears revealed Cryptobia acipenseris and Haemogregarina acipenseris infection. Developmental stages of the coccidia Goussia acipenseris Molnár, 1986 and Goussia vargai Molnár, 1986 were frequently seen in the intestine of the dissected fish.     

A molecular approach to detect hybridisation between crucian carp (Carassius carassius) and non-indigenous carp species (Carassius spp. and Cyprinus carpio)

1. Releases of non‐native fish into the wild is an increasing problem posing considerable ecological and genetic threats through direct competition and hybridisation. 2. We employed six microsatellite markers to identify first generation hybrids and backcrosses between native crucian carp (Carassius carassius) and introduced goldfish (C. auratus) and common carp (Cyprinus carpio) in the U.K. We also investigated the genetic characteristics of the taxonomically controversial gibel carp (Carassius spp.) from sites across Europe. 3. Natural hybridisation between goldfish and crucian carp occurs frequently, although hybrids between all other species pairs were observed. Only 62% of British crucian carp populations (n = 21) consisted exclusively of pure crucian carp. In some populations hybrids were so frequent, that no pure crucian carp were caught, indicating a high competitive ability of hybrids. 4. Most hybrids belonged to the F1 generation but backcrossing was evident at a low frequency in goldfish × crucian carp hybrids and goldfish × common carp hybrids. Furthermore, some local populations had high frequencies of backcrosses, raising the opportunity for introgression. 5. Gibel carp from Germany and Italy belonged to two triploid clonal lineages that were genetically closely related to goldfish, whereas all individuals identified from British populations proved to be crucian carp × goldfish hybrids. 6. Our study suggests that the release of closely related exotic cyprinids not only poses a threat to the genetic integrity and associated local adaptations of native species, but may also contribute to shifts in community structure through competitive interactions.     

POLYPHASIC CHARACTERIZATION OF DOLICHOSPERMUM SPP. AND SPHAEROSPERMOPSIS SPP. (NOSTOCALES,CYANOBACTERIA): MORPHOLOGY, 16S rRNA GENE SEQUENCES AND FATTY ACID AND SECONDARY METABOLITE PROFILES1

The genera Dolichospermum (Ralfs ex Bornet et Flahault) Wacklin, L. Hoffm. et Komárek and Sphaerospermopsis Zapomělová, Jezberová, Hrouzek, Hisem, K. ?eháková et Komárk.‐Legn. represent a highly diversified group of planktonic cyanobacteria that have been recently separated from the traditional genus Anabaena Bory ex Bornet et Flahault. In this study, morphological diversity, phylogeny of the 16S rRNA gene, production of fatty acids, and secondary metabolite profiles were evaluated in 33 strains of 14 morphospecies isolated from the Czech Republic. Clustering of the strains based on 16S rRNA gene sequences corresponded to wider groups of species in terms of morphology. The overall secondary metabolite and fatty acid profiles, however, were not correlated to each other and neither were they correlated to the 16S rRNA phylogeny nor the morphology of the strains. Nevertheless, a minor part of the detected secondary metabolites (19% of all compounds) was present only in close relatives and can be thus considered as autapomorphic features.     

Morphological features of an endangered Japanese strain of Cyprinus carpio: reconstruction based on seven SNP markers

Morphological analyses of 183 specimens of Japanese common carp Cyprinus carpio (171 from Lake Biwa and 12 from nursery ponds) using genetic hybrid indices demonstrated that the typical native Japanese strain of C. carpio has a more elongate body, more branched dorsal‐fin rays, fewer and shorter gill rakers, more developed pneumatic bulb, more coiled pneumatic duct, longer posterior swimbladder and shorter intestine than the typical introduced C. carpio. These results provide a basis for a better understanding of the ecological characteristics and taxonomic status of the endangered Japanese strain of C. carpio.     

Effect of total dissolved gas supersaturation on the survival of common carp (Cyprinus carpio) and silver carp (Hypophthalmichthys molitrix)

Flood discharge results in total dissolved gas (TDG) supersaturation downstream of a dam during the flood period. Fish suffer death from gas bubble disease (GBD) caused by TDG supersaturation. Nonetheless, current studies mainly attach importance to the survival of benthic fish affected by TDG supersaturation in the Yangtze River in China. Few studies have attempted to investigate the survival of pelagic fish influenced by TDG supersaturated water and compare the tolerance characteristics to TDG supersaturation between benthic and pelagic fish. To identify the survival of fish species that inhabit the various water layers affected by TDG supersaturation, silver carp (Hypophthalmichthys molitrix) (pelagic fish) and common carp (Cyprinus carpio) (benthic fish) were chosen to conduct an acute exposure experiment of four different TDG supersaturation levels (125%, 130%, 135% and 140%). The findings illustrated that the two fish species both exhibited evident aberrant behaviours of maladjustment in TDG supersaturated water. Obvious GBD symptoms were also found in the test fish. The survival probability of silver carp and common carp decreased with increasing levels of TDG supersaturation. The median survival time (ST₅₀) values of the silver carp exposed to four levels of TDG supersaturated water (125%, 130%, 135% and 140%) were 26.84, 7.96, 5.56 and 3.62 h, respectively, whereas the ST₅₀ values of common carp were 53.50, 26.00, 16.50 and 11.70 h, respectively. When compared with common carp, silver carp had a weaker tolerance to TDG-supersaturated water and were vulnerable to GBD. It shows that levels above 125% are not safe for common carp survival. In terms of the tolerance threshold value, silver carp merits further investigation because it showed lower tolerance to TDG than did common carp.     

Genetic characterization of common carp (Cyprinus carpio) populations from Greece using mitochondrial DNA sequences

Wild common carp from two lakes and two rivers in Greece were genetically characterized with sequencing analysis of two mitochondrial DNA segments: cytochrome b (1119 bp) and D-loop (646 bp). A total of 9 variable singleton sites and 7 unique haplotypes were detected. A common haplotype was found in three out of the four populations examined, which seems to be the ancestral one and represents the European origin of common carp from Greece. This haplotype could be also justified by the introductions reported with individuals belonging to the Central European race, into many natural habitats in Greece. Limited genetic variation — in Evros and Aliakmonas populations — could be due to bottleneck effects and small effective population sizes, whereas the different haplotypes found in Lake Volvi could represent different common carp stocks. Values of sequence divergence among Greek haplotypes ranged from 0.0006 to 0.0023. The Neighbour-Joining (NJ) phylogenetic tree constructed based on the combined sequences, reveals that the populations of common carp from Greece belong to the European group of populations — which is highly divergent from the South East-Asia cluster — and to the subspecies Cyprinus carpio carpio.     

Polymorphism of microsatellite markers in Russian common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.) breeds

Using five microsatellite loci, genotyping and genetic diversity estimates were obtained for nine samples representing seven common carp breeds most widespread in Russia. For comparison, the samples of Amur wild common carp (Cyprinus carpio haematopterus) and a sample of European Hungarian carp were used. In the samples examined (n = 148) a total of 78 alleles were revealed. The highest mean allele number per locus (7.3) was identified in Amur wild common carp, while the lowest number was found in Cherepets carps (4.0). In different breeds, the observed heterozygosities varied from 0.819 (Altai carp) to 0.651 (Cherepets scaly carp). Three out of five microsatellite loci (MFW-24, MFW-28, and MFW-19) revealed a high level of population differentiation. In the dendrogram of genetic differences, all breeds clustered into two groups. One of these groups was composed of the two strains of Ropsha carp, Stavropol carp, Amur wild common carp, and the two samples of Cherepets carp. The second cluster included Altai carp (Priobskii and Chumysh populations), two Angelinskii carp breeds (mirror and scaly), and Hungarian carp. The pairs of breeds/populations/strains, having common origin, were differentiated. Specifically, these were two populations of Altai carp, two strains of Ropsha carp, as well as the breeds of Angelinskii and Cherepets carps. The reasons for genetic differentiation of Russian common carp breeds, as well as the concordance of the evolutionary histories of these breeds, some of which originated from the European breeds, while the others contain substantial admixture of the Amur wild common carp, are discussed.     

PKX, the causative agent of proliferative kidney disease (PKD) in Pacific salmonid fishes and its affinities with the Myxozoa

Proliferative kidney disease (PKD), caused by an unclassified protozoan (PKX), is reported from Pacific salmon, Oncorhynchus tshawytscha (Walbaum) and O. kisutch (Walbaum), and steelhead trout, Salmo gairdneri Richardson, held at the Mad River Hatchery in California, USA. The cumulative mortality attributed to the disease was 95, 13, and 18% respectively. The mortalities were greatest at mean water temperatures of 12-14 degrees C during July 1983. The ultrastructure of the PKX organism and its associated pathology during clinical disease in all three species were consistent with those of the parasite in rainbow trout (Salmo gairdneri) as described in European outbreaks. Significant mortalities did not occur after August, at which time the parasite could no longer be detected in the salmon species. The steelhead continued to exhibit parasites in the kidney interstitium and epithelium and lumens of the tubules. Myxosporidan trophozoites and developing spores were also observed in the lumens of the kidney tubules of these fish. Although a mixed infection with another parasite may have occurred, evidence suggests that the myxosporidans are later stages of PKX. They were only observed in fish exposed to water with the infective stage and were particularly prominent in recovering fish. The PKX organism is similar to UBO, an unclassified protozoan of carp suspected to be an early stage of Sphaerospora renicola Dykovà & Lom. Both parasites infect the blood and kidney, divide by endogeny, and are released by disintegration of the primary mother cell. The intraluminal myxosporean forms show similarities to Sphaerospora spp. in that they are monosporous and sporoblasts are formed within pseudoplasmodia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polymorphism of the cytochrome oxidase b gene (<Emphasis Type="Italic">cyt b</Emphasis>) in Russian populations of common carp and wild common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Polymorphism of the mitochondrial cyt b gene was examined in 35 individuals of common carp and wild common carp (Cyprinus carpio L.). The fish examined represented two natural populations from Khabarovsk krai (Ac and Am), Volga wild common carp, Don wild common carp, and two common carp breeds, Ropsha (strains BB and MM) common carp and Hungarian common carp. The highest level of nucleotide (π) and haplotype (h) diversity was detected in two strains of Ropsha common carp (MM, π = 0.67%, h = 0.7; and BB, π = 0.21%, h = 0.9) and in one population (Am) of Amur wild common carp (π = 0.26%; h = 0.6). The second population of Amur wild common carp (Ac) and Hungarian common carp were characterized by lower variation estimates (π = 0.035%, h = 0.4; and π = 0.09%, h = 0.7, respectively). Genetic homogeneity was demonstrated for the populations of Volga and Don wild common carp (π = 0, h = 0). In the sample of the cyt b sequences examined, three lineages were identified. Lineages I and II united all haplotypes of the Am Amur wild common carp along with two haplotypes of Ropsha common carp, strain MM. The third lineage (III) was formed by the haplotypes of three individuals of Ropsha common carp strain MM, all representatives of Ropsha common carp strain BB, Hungarian common carp, Ac Amur wild common carp, and Don and Volga wild common carps. Statistically significant amino acid differences were observed only for the sequences, corresponding to haplotypes of lineage III, and the sum of sequences of lineages I and II. Effectiveness of different types of markers to differentiate the two subspecies of European and Amur wild common carp (C. c. carpio and C. c. haematopterus) is discussed, as well as the issues of the origin and dispersal of Russian common carp and wild common carp breeds.     

Vegetalising factor extracted from the fish swimbladder and tested on presumptive ectoderm ofTriturus embryos

Summary Vegetalising factor was isolated from swimbladder of crusian carp (Carassius auratus) by solubilishing with 8 M urea the precipitate obtained after digesting the swimbladder with collagenase. The urea-soluble fraction vegetalised isolated presumptive ectoderm ofTriturus gastrula and produced both undifferentiated mesodermal and endodermal cells. Brief heating of the fraction changed its capacity to produce organised mesodermal tissues, such as notochord and somite, and the frequency of induction of undifferentiated cells was reduced. By inserting the urea-soluble fraction into the blastocoel of an early gastrula, embryos without epidermis were obtained. Some of the embryos consisted of undifferentiated mesodermal and endodermal cells, but in the remaining ones small fragments of notochord, small numbers of somites and pronephros developed, enclosed by endodermal cells.     

Efficacy of Allicin in Decreasing Lead (Pb) Accumulation in Selected Tissues of Lead-Exposed Common Carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>)

The objective of this study was to evaluate the effects of allicin, the main biologically active component of garlic clove extracts, on lead levels in different common carp tissues including liver, kidney, brain, bone, and blood following experimental lead poisoning. Fish were divided randomly into five groups depending on the combination of lead acetate and allicin treatments. Lead acetate exposure (7.0 mgL⁻¹, 10 days) caused a significant increase in mean Pb concentrations in all examined tissues in comparison to control unexposed fish (p < 0. 001). The results showed that allicin supplementation is effective in decreasing lead accumulation in all examined tissues of common carp. The promising ameliorative effects of allicin on tissue lead levels of common carp make it a good candidate for therapeutic intervention of lead poisoning. However, more studies are required to elucidate the pharmacokinetic effects of allicin and also molecular basis of the ameliorative properties of allicin in lead poisoning.     

Outbreaks of phaeohyphomycosis in the chinook salmon (Oncorhynchus tshawytscha) caused by Phoma herbarum

Phoma herbarum has been associated with two outbreaks of systemic mycosis in hatchery-reared chinook salmon (Oncorhynchus tshawytscha) fingerlings. Affected fish exhibited abnormal swimming behavior, exophthalmia, multiple rounded areas of muscle softening, protruded hemorrhagic vents, and abdominal swelling. In all affected fish, swimbladders were filled with whitish creamy viscous fungal mass, surrounded by dark red areas in swimbladder walls, kidneys, and musculature. Clinical and histopathological examinations suggest that the infection may have started primarily in the swimbladder and then spread to the kidneys, gastrointestinal tract, and surrounding musculature. Consistent microscopical findings included broad septate branched fungal hyaline hyphae, 5–12 μm in diameter within the swimbladder, stomach, and often within and adjacent to blood vessels. Profuse growths of woolly brown fungal colonies were obtained from swimbladders and kidneys on Sabouraud medium. On corn meal agar the formation of pycnidia, characteristic of Phoma spp., was detected within 10 days of incubation. Morphological and molecular analyses identified this fungus as Phoma herbarum. This report underscores systemic fungal infections as a threat to raceway-raised salmon.