The in vitro antifungal activity of 30 Chinese herb extracts to Saprolegnia sp.

Chinese traditional medicines have been used for several thousands of years in Asian countries, not only in humans but also in many animal species. These compounds prevent and control different types of diseases including internal diseases as well as some infectious diseases, where the aetiological agent is viral, bacterial, parasitic or mycotic. Rhizoma coptidis is believed to inhibit Shigella dysenteriae and that Radix isatidis can prevent flu caused by the influenza virus. It is thus hypothesized that some of these traditional herbal compounds will have anti‐fungal activity. Saprolegniosis is a disease common in fish and their eggs in both fresh and brackish water; a newer, safer medication against Saprolegnia is needed after the prohibition in many countries of the extremely effective fungicide, malachite green. In the present study an attempt is made to identify herbal compounds that have anti‐Saprolegnia activity. A strain of Saprolegnia, CCF1301, was isolated from the skin of infected grass carp (Ctenopharyngodon idella) and identified as Saprolegnia ferax by the 26S rDNA D1/D2 region and ITS region. This strain was used to evaluate the antifungal activity of thirty Chinese traditional herbal medicine extracts, and a modified dish dilution method was developed for the evaluation. Saprolegnia‐infected rapeseeds with visible hyphae were transplanted onto prepared PDA plates containing 2 g L^?1 herbal plant extracts and incubated at 20°C for 48 h. Each herbal plant species was tested in triplicate. Those herbal plant extracts that showed negative mycelium presence at 2 g L^?1 were further tested for minimum inhibitory concentration (MIC) evaluation. The results showed that Syzygium aromaticum, Magnolia officinalis, Melaphis chinensis, Euphorbia fischeriana Steud, and Sophora flavescentis exhibited enhanced growth inhibition at 2 g L^?1 and MIC values of 500, 62.5, 250, 62.5, 250 mg L^?1 concentrations, respectively. It was obvious that Magnolia officinalis and Euphorbia fischeriana Steud exhibited the best antifungal activity. Since there is a high natural toxicity in Euphorbia fischeriana Steud, its applicability as the main ingredient in an aquaculture therapeutic formulation requires further research. Thus, Magnolia officinalis would appear to be the more valuable antifungal herbal species with which to pursue further research.     

Molecular identification of a bronopol tolerant strain of Saprolegnia australis causing egg and fry mortality in farmed brown trout, Salmo trutta

Some species of the genus Saprolegnia, such as Saprolegnia diclina and Saprolegnia ferax are responsible for devastating infections on salmonid eggs. Members of this group cause saprolegniasis, a disease resulting in considerable economic losses in aquaculture. Although both S. diclina and S. ferax have received much attention, the role of other Saprolegnia species in infecting fish eggs is less known. For this purpose, we have investigated the aetiology of chronic egg mortality events occurring in farmed brown trout, Salmo trutta. A total of 48 isolates were obtained from eggs with signs of infection as well as from water samples. A molecular analysis based on nrDNA internal transcribed spacer (ITS) operational taxonomic units indicated that the majority of the isolates correspond to Saprolegnia australis. All isolates of S. australis exhibited the same random amplified polymorphic DNA (RAPD) band patterns suggesting that a single strain is implicated in egg infections. The isolates followed Koch postulates using trout eggs and fry. Under standard concentrations of bronopol commonly used in farms, these isolates could grow, but not sporulate. However, both growth and sporulation were recovered when treatment was removed. This study shows that S. australis can infect and kill salmon eggs, and helps in defining oomycetes core pathogens.     

Genetic structure of silver Prussian carp <Emphasis Type="Italic">Carassius</Emphasis> (Superspecies <Emphasis Type="Italic">auratus</Emphasis>) (Linnaeus, 1758) colonies from the Middle Dnieper Basin

Population genetic structure of silver Prussian carp from the Middle Dnieper basin was established by means of biochemical gene marking and cytometric analysis. In addition to diploid goldfish Carassius auratus, which was the basic species of bisexual colonies, a number of biotypes of triploid silver Prussian carp C. gibelio were identified. These biotypes either formed isolated unisexual populations, or were an admixture to the bisexual colonies. In addition, in bisexual colonies, triploid females of non-clonal origin were identified. Despite the fact that all forms of C. gibelio reproduced by means of gynogenesis, some of them (C. gibelio-1) formed isolated unisexual populations, while the others (C. gibelio-2, -3, and -4) usually coexisted with C. auratus.     

Effect of Temperature on the Morphometrical and Physical Parameters of Erythrocytes and Polymorphonuclear Leucocytes in <Emphasis Type="Italic">Carassius gibelio</Emphasis> (Bloch)

Dynamics of the morphometric and physical properties of hemocytes of the Prussian carp Carassius gibelio (Bloch) under the influence of a temperature factor has been studied with atomic force microscopy in experiments in vitro. It is found that, at a low incubation temperature (5°C), as opposed to room temperature (20°C), morphometric parameters change in erythrocytes; at a high temperature (40°C) they change in polymorphonuclear leucocytes. The low incubation temperature reduces the adhesion and elasticity of polymorphonuclear leucocytes and erythrocytes of C. gibelio, whereas a high incubation temperature leads to a decrease in adhesion in polymorphonuclear white blood cells.     

Isolation and characterization of microsatellite markers from Fangzheng silver crucian carp,Carassius auratus gibelio (Bloch), and cross‐amplification in the closely related species crucian carp,Carassius auratus auratus (Linnaeus)

We report the development of 20 microsatellite markers for Fangzheng silver crucian carp, Carassius auratus gibelio (Bloch). Nineteen out of 20 showed polymorphism with alleles ranging from two to 14. These loci were screened to amplify the closely related species crucian carp, Carassius auratus auratus (Linnaeus) and all of them can amplify DNA with the size similar to the former. The origin of silver crucian carp is in issue and the population genetic structure is still unclear. Microsatellite markers isolated from the silver crucian carp and their utility in the crucian carp will be useful for these researches.     

Length–weight relationships of an invasive cyprinid fish (Carassius gibelio) from 12 Greek lakes in relation to their trophic states

The length–weight relationships of the cyprinid fish Carassius gibelio (Bloch, 1782) are described for the 12 most important lakes of Greece. Carassius gibelio is an allogynogenetic fish species, which was introduced into Greek lakes during the late 1970s. The values of the exponent b of the length–weight relationships ranged from 2.33 to 3.38, and varied with the trophic state of the lake. In eutrophic lakes these b values were significantly (P < 0.001) lower than in oligotrophic or mesotrophic lakes. Relationships between the b values and phosphorus concentrations were logarithmic: b = 1.37–0.13 log (PO₄‐P); total length, fork length and standard length were linear (in all cases: r² > 0.95). In most of the lakes, populations consisted of females, with the exception of Pamvotis, Doirani and Koronia lakes where a small proportion were males.     

Influence of environmental parameters on growth pattern and population structure of Carassius auratus gibelio in Eastern Ukraine

Population structure and growth parameters of Prussian carp (Carassius auratus gibelio) were studied in 12 freshwater ecosystems of the Donbass region (Eastern Ukraine). These ecosystems differed significantly with respect to their surface area, water transparency and annual concentrations of phosphorus and nitrogen. Amongst the studied ecosystems, diploid males and females as well as a smaller percentage of triploid females were found. The population structure of C. a. gibelio varied significantly in terms of the percentage of triploids and sex ratio amongst diploids. A considerable proportion of triploid females (>10%) was found in four ecosystems with intermediate surface area (38–50 ha) and relatively high growth rate of specimens. The sex ratio amongst diploids was significantly female-biased in seven of 12 ecosystems, including those where triploids were present in considerable numbers. The growth parameters of Prussian carp were significantly correlated with ecosystem characteristics, but the growth patterns of diploids and triploids were not significantly different from each other. The restricted distribution of triploid specimens suggests that the triploid form might counter diverse environmental challenges, whereas the diploid form of Prussian carp seems to be efficiently adapted to a wide range of ecosystem conditions.     

Reprint of: Saprolegnia strains isolated from river insects and amphipods are broad spectrum pathogens

Saprolegnia species are destructive pathogens to many aquatic organisms and are found in most parts of the world. Reports based on phylogenetic analysis suggest that Saprolegnia strains isolated from aquatic animals such as crustaceans and frogs are close to Saprolegnia strains isolated from infected fish or fish eggs and vice versa. However, it has often been assumed that host specificity occurs for each individual isolate or strain. Here we demonstrate that Saprolegnia spp. can have multiple hosts and are thus capable of infecting different aquatic organisms. Saprolegnia delica, Saprolegnia hypogyna, and 2 strains of Saprolegnia diclina were isolated from aquatic insects and amphipods while S. delica, Saprolegnia ferax, Pythium pachycaule, and a Pythium sp. were isolated from the water of a medium to fast flowing river. The ITS region of the rRNA gene was sequenced for all isolates. In challenge experiments, all four isolates from insects were found to be highly pathogenic to eggs of Atlantic salmon (Salmo salar) and embryos of the African clawed frog (Xenopus laevis). We found that Saprolegnia spp. isolated from salmon eggs were also able to successfully establish infection in nymphs of stonefly (Perla bipunctata) and embryos of X. laevis). These results suggest that Saprolegnia spp. are capable of infecting multiple hosts, which may give them an advantage during seasonal variation in their natural environments.     

Morphological and molecular phylogenetic analysis of two Saprolegnia sp. (Oomycetes) isolated from silver crucian carp and zebra fish

Two Saprolegnia isolates, JY isolated from silver crucian carp (Carassius auratus gibelio Bloch) and BMY isolated from zebra fish (Brachydanio rerio Hamilton) came from infections occurring concurrently in different locations in China. To confirm whether the two isolates were from the same Saprolegnia clone, comparative studies have been carried out based on their morphological, physiological and molecular characteristics. Observations showed that morphologically (both asexual and sexual organs) the two isolates were broadly similar and both isolates underwent repeated zoospore emergence. Comparing 704 base pairs of internal transcribed spacer (ITS) region and the 5.8S rDNA, we found isolates JY and BMY shared an identical ITS sequence with a minor variation (99.6 % similarity). Forty available sequences for representatives Saprolegnia spp. belonged to four phylogenetically separate clades. The two studied isolates fell within clade I that comprised a group of isolates which showed almost an identical ITS sequence but had been identified as a number of different morphological species. Our findings suggest that isolates JY and BMY appear to belong to the S. ferax clade and this clade (I) contains a number of closely related phylogenetic species. This is distinct from the more common fish pathogenic isolates, which belong to the S. parasitica clade (III) and are characterized by having cysts decorated by bundles of long hooked hairs and two further clades (II and IV) containing largely saprotrophic or soil born species.     

Polymorphic microsatellites from silver crucian carp (Carassius auratus gibelio Bloch) and cross‐amplification in common carp (Cyprinus carpio L.)

Fifteen polymorphic microsatellites were isolated from the genome of silver crucian carp (Carassius auratus gibelio Bloch). Allele numbers ranged from two to four with an average of 2.7/locus, and the proportion of tri‐ and diallelic heterozygotes was 99.3%. The individuals tested seem to have originated from two different clonal lines: 14 of 16 showed the same genotype at all loci tested, whereas the remaining two were also identical, but different from the former ones. Eleven out of 15 primer pairs cross‐amplified products from the genome of common carp, whereas only five from that of zebrafish.     

Behavioural pattern in cyprinid fish below a weir as detected by radio telemetry

Changes in the behaviour of cyprinids belonging to different ecology groups were observed below a weir that was restricting their upstream migrations during the spawning period May–July. Positions of chub Leuciscus cephalus (L.), Prussian carp Carrasius auratus gibelio (Bloch 1783), bream Abramis brama (L.) and white bream Blicca bjoerkna (L.) were recorded every 12 min over a 24‐h period using an automatic data logger (ALS) in the upper Elbe River, Czech Republic. For all observed species, two activity peaks occurred; however, the interspecies timing of these peaks differed. Peak times for chub, bream and white bream occurred at night and during daylight, whereas Prussian carp was active mostly at night. Specimens belonging to the rheophilous group suggested more efficient behavioural tactics in passing the weir, staying below the obstacle one month longer than did eurytopic species.     

Saprolegnia strains isolated from river insects and amphipods are broad spectrum pathogens

Saprolegnia species are destructive pathogens to many aquatic organisms and are found in most parts of the world. Reports based on phylogenetic analysis suggest that Saprolegnia strains isolated from aquatic animals such as crustaceans and frogs are close to Saprolegnia strains isolated from infected fish or fish eggs and vice versa. However, it has often been assumed that host specificity occurs for each individual isolate or strain. Here we demonstrate that Saprolegnia spp. can have multiple hosts and are thus capable of infecting different aquatic organisms. Saprolegnia delica, Saprolegnia hypogyna, and 2 strains of Saprolegnia diclina were isolated from aquatic insects and amphipods while S. delica, Saprolegnia ferax, Pythium pachycaule, and a Pythium sp. were isolated from the water of a medium to fast flowing river. The ITS region of the rRNA gene was sequenced for all isolates. In challenge experiments, all four isolates from insects were found to be highly pathogenic to eggs of Atlantic salmon (Salmo salar) and embryos of the African clawed frog (Xenopus laevis). We found that Saprolegnia spp. isolated from salmon eggs were also able to successfully establish infection in nymphs of stonefly (Perla bipunctata) and embryos of X. laevis). These results suggest that Saprolegnia spp. are capable of infecting multiple hosts, which may give them an advantage during seasonal variation in their natural environments.     

Betrachtungen an Schädeln von Goldfischen (Carassius gibelio f. auratus)

Studies on skulls of goldfishes (Carassius gibelio f. auratus) The aim of the study is to analse skulls of some races of goldfish, the domesticated form of Carassius gibelio Bloch, 1782. The problem: are changes of these skulls parallelisms to skulls of other domesticated animals originating from different species. Parallelism is a most interesting phenomenon of phylogeny abundant but not regular, very frequent in domesticated animals. Skulls of normal goldfish, of frinetail and tigerhead have been compared. In total the skulls of fringetail and especially tigerhead are broader, higher, stouter, the faceart shortened. Remarkable in detail: the opercula are slender and higher in fringetail and tigerhead than in normal goldfish, the preopercula shortened and their ventral art bended upwards, influencin the position of the mouth. In comparison with normal goldfish the skulls of fringetail and tigerhead have to be classified als parallelisms to skulls of some races of other domesticated secies. Finally: some remarks on parallelism, convergence and pluripotenz and agout modern viewpoints on the relations between genes and complex organismal patterns.     

Effects of sodium chloride, formalin and iodine on the hatching success of common carp, Cyprinus carpio, eggs

Saprolegniales are ubiquitous in natural water supplies of fish hatcheries, and often cause serious disease problems. Sodium chloride, formalin and iodine, administered twice a day as a flush at different concentrations, were tested on infected eggs of common carp, Cyprinus carpio, to evaluate their antifungal activity and effect on hatching rates. Sodium chloride at 35 000 mg L^?1 and formalin at 400 mg L^?1 were found to be most effective in controlling Saprolegnia sp., with 85.4 and 91.8% hatching rates, respectively. Iodine increased the hatching rate by 27% at 200 mg L^?1 (P < 0.05). There were infections on eggs exposed to all levels of iodine, but not on eggs treated with sodium chloride and formalin. Sodium chloride and formalin were more effective than iodine in controlling Saprolegnia. Sodium chloride is a safe, efficacious and economical treatment of Saprolegniosis and is therefore recommended for treating common carp eggs.     

Biological traits of rare males in the population of Carassius gibelio (Actinopterygii: Cyprinidae) from Lake Pamvotis (north‐west Greece)

The gynogenetic population of Prussian carp Carassius gibelio in Lake Pamvotis (north‐west Greece) included a small percentage of males (2–3%). Cytogenetic analysis revealed heterogeneity of the population at the ploidy level: all females were triploid (modal chromosome number 156–162 chromosomes) while the males were either triploid (modal number 156–158 chromosomes) or tetraploid (modal number 200–214 chromosomes). Morphometric analysis revealed significant differences in body shape among these three groups. The males were more slender than females, while the triploid males could be discriminated from the tetraploid by the morphology of their caudal peduncle. The males attained a significantly lower asymptotic standard length (L_S) (25·04 cm) than females (29·75 cm) and had lower exponent b of the L_S and mass relationship. Under laboratory conditions, intraspecific crossings of C. gibelio males with females could give viable all‐female offspring; in comparison with goldfish Carassius auratus males, the fertility of the C. gibelio males was generally reduced but remained highly variable among individuals.     

A Set of Polymorphic Trinucleotide and Tetranucleotide Microsatellite Markers for Silver Crucian Carp (Carassius auratus gibelio) and Cross-Amplification in Crucian Carp

Silver crucian carp (Carassius auratus gibelio Bloch), as a gynogenetic fish, is a promising model for the study of the evolutionary genetics of vertebrates. We have developed 59 polymorphic trinucleotide and tetranucleotide markers for the silver crucian carp through the biotin capture method and radioactive-labeling hybridization. The number of alleles ranged from 2 to 12 in the population, and the average proportion of heterozygotes (including tri- and diallelic) at polymorphic loci was 76.8%. In addition, these loci were successfully applied to a close relative, the crucian carp (Carassius auratus), by cross-amplification, as shown by the range of alleles (2–19), observed heterozygosity (0.1765–0.9706), expected heterozygosity (0.2392–0.9421), and polymorphism information content (0.2186–0.9236).     

Diversity of Aquatic Pseudomonas Species and Their Activity against the Fish Pathogenic Oomycete Saprolegnia

Emerging fungal and oomycete pathogens are increasingly threatening animals and plants globally. Amongst oomycetes, Saprolegnia species adversely affect wild and cultivated populations of amphibians and fish, leading to substantial reductions in biodiversity and food productivity. With the ban of several chemical control measures, new sustainable methods are needed to mitigate Saprolegnia infections in aquaculture. Here, PhyloChip-based community analyses showed that the Pseudomonadales, particularly Pseudomonas species, represent one of the largest bacterial orders associated with salmon eggs from a commercial hatchery. Among the Pseudomonas species isolated from salmon eggs, significantly more biosurfactant producers were retrieved from healthy salmon eggs than from Saprolegnia-infected eggs. Subsequent in vivo activity bioassays showed that Pseudomonas isolate H6 significantly reduced salmon egg mortality caused by Saprolegnia diclina. Live colony mass spectrometry showed that strain H6 produces a viscosin-like lipopeptide surfactant. This biosurfactant inhibited growth of Saprolegnia in vitro, but no significant protection of salmon eggs against Saprolegniosis was observed. These results indicate that live inocula of aquatic Pseudomonas strains, instead of their bioactive compound, can provide new (micro)biological and sustainable means to mitigate oomycete diseases in aquaculture.     

Life history characteristics of an invasive cyprinid fish (Carassius gibelio) in Chimaditis Lake (northern Greece)

The life history characteristics of an invasive cyprinid fish, the Prussian carp (Carassius gibelio) were examined in Lake Chimaditis (northern Greece). The population is dominated by females that are able to reproduce gynogenetically. Fish samples were collected using trammel nets from August 2004 to July 2005. Their total length (TL) ranged between 21.9 and 37.0 cm. The length–weight relationship was W = 0.0336TL^2.81 (r² = 0.92, n = 600) and the mean Fulton condition factor exhibited monthly variation from 0.172 (May) to 0.186 (August). According to the annual growth marks present, the lifespan of the Prussian carp in Lake Chimaditis is 6 years. The growth parameters were estimated as L_∞ = 34.46 cm, K = 0.297 year^?1, t₀ = ?1.994 year. Absolute fecundity (F_A) ranged between 26 000 and 176 600 oocytes (mean = 66990) and relative fecundity between 78 and 251 oocytes per gram of total weight (mean = 158). Absolute fecundity increased exponentially with length (F_A = 0.286 × TL^3.66, r² = 0.66) and weight (F_A = 17.93 × W^1.36, r² = 0.73) of the fish.     

Boric Acid Inhibits Germination and Colonization of Saprolegnia Spores In Vitro and In Vivo

Saprolegnia infections cause severe economic losses among freshwater fish and their eggs. The banning of malachite green increased the demand for finding effective alternative treatments to control the disease. In the present study, we investigated the ability of boric acid to control saprolegniosis in salmon eggs and yolk sac fry. Under in vitro conditions, boric acid was able to decrease Saprolegnia spore activity and mycelial growth in all tested concentrations above 0.2 g/L, while complete inhibition of germination and growth was observed at a concentration of 0.8 g/L. In in vivo experiments using Atlantic salmon eyed eggs, saprolegniosis was controlled by boric acid at concentrations ranging from 0.2–1.4 g/L during continuous exposure, and at 1.0–4.0 g/L during intermittent exposure. The same effect was observed on salmon yolk sac fry exposed continuously to 0.5 g/L boric acid during the natural outbreak of saprolegniosis. During the experiments no negative impact with regard to hatchability and viability was observed in either eggs or fry, which indicate safety of use at all tested concentrations. The high hatchability and survival rates recorded following the in vivo testing suggest that boric acid is a candidate for prophylaxis and control of saprolegniosis.     

Susceptibility of Prussian carp infected by metacercariae of Posthodiplostomum cuticola (v. Nordmann, 1832) to fish predation

The susceptibility of prey fish infected by metacercariae of Posthodiplostomum cuticola (Digenea: Diplostomatidae) to the predation of non-host predators under experimental conditions was investigated. Parasitized young-of-the-year Prussian carp Carassius auratus were consumed significantly more often by perch Perca fluviatilis compared to non-parasitized individuals, independent of Prussian carp density. The proportion of parasitized and non-parasitized fish consumed by the predator remained stable at four different prey densities. The probability of predation did not increase with the intensity of parasite infection. The effect of P. cuticola on the host seems to result mostly from pathological changes (poor condition, black spots). However, our results provide evidence of higher probability of parasitized Prussian carp being consumed by fish predators under experimental conditions.