Fungi isolated from cultured eggs, alevins and broodfish of brown trout in a hatchery affected by saprolegniosis

The aquatic fungi cultured from eggs, alevins and broodfish of brown trout Salmo trutta belonged to the genus Saprolegnia and were identified as S. diclina , S. australis , S. ferax , S. furcata , S. hypogyna , S. unispora and S. parasitica . The species obtained from infected eggs and alevins were different to those from infected fish. Several Saprolegnia species were isolated from eggs and alevins, whereas all the isolates obtained from broodfish were the pathogenic S. parasitica .     

Re-evaluation of the enigmatic species complex Saprolegnia diclina-Saprolegnia parasitica based on morphological, physiological and molecular data

The phylogenetic relationships among isolates of the Saprolegnia diclina-Saprolegnia parasitica complex were investigated based on ITS rDNA sequences, and correlated with morphological and physiological characters. The isolates studied belong to five phylogenetically separate clades. The majority of presumed parasitic isolates, mostly isolated from fish lesions, fell within a clade that comprises isolates which has been variously named as S. diclina Type 1, S. parasitica, Saprolegnia salmonis or just as unnamed Saprolegnia sp. Presence of bundles of long-hooked hairs on secondary cysts, high frequency of retracted germination, and oogonia production at 7 degrees C (when occurring) were characteristic of this clade. A single isolate identified as S. diclina Type 2 clustered in a clade along with Saprolegnia ferax isolates. The isolates identified as S. diclina s. str. (S. diclina Type 3) distributed in two clades and appeared closely related to Saprolegnia multispora and to a number of Chilean isolates identified as Saprolegnia australis. The ITS sequences of clade I were almost identical even though the isolates were of diverse geographical origins and showed physiological and morphological differences and variations in their pathogenicity. This suggest these species reproduces clonally even in apparently sexually competent isolates. Adaptation to parasitism in Saprolegnia might have occurred at spore level by the development of long-hooked hairs to facilitate host attachment and selection of a retracting germination. The use of the name S. parasitica should be assigned to isolates of clade I that contained isolates forming cysts with bundles of long-hooked hairs.     

Characterisation of Saprolegnia sp. isolates from channel catfish.

Nineteen channel catfish isolates of Saprolegnia sp. obtained from 5 separate fish farms in Mississippi, which became affected by winter kill syndrome during 1991 and 1996, were investigated with respect to physiological characteristics and genetic variation. Isolates of S. parasitica from crayfish and S. diclina were included for comparison. Most strains of catfish isolates grew well at 20 and 30 degrees C. Repeated zoospore emergence was found in catfish isolates of Saprolegnia sp. and S. parasitica, but not in S. diclina. Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) was applied for a further characterisation of the isolates. The RAPD analysis among Saprolegnia spp. isolates was constructed from 686 amplified products in 67 separable positions and indicated that the catfish isolates of Saprolegnia sp. are composed of 3 genetically distinct groups.     

Bacterial skin flora variation and in vitro inhibitory activity against Saprolegnia parasitica in brown and rainbow trout

Variations in the number and diversity of bacteria from the skin of brown trout Salmo trutta L. and rainbow trout Oncorhynchus mykiss Walbaum were surveyed from different rivers and fish farms in northern Spain. In addition to determining bacterial populations in skin samples of healthy fish, bacterial populations were determined from skin lesions (of brown trout only) infected with Saprolegnia parasitica, the causal agent of saprolegniosis. Mean bacterial counts from skin lesions of brown trout suffering from saprolegniosis were nearly 1000 times greater than from the skin of uninfected brown and rainbow trout. More than 20 different genera of bacteria were identified, with isolates of Aeromonas and Iodobacter being the predominant genera associated with saprolegniosis lesions. The in vitro inhibitory activity of 72 of these skin isolates was tested against S. parasitica using 3 different assays. These included (1) assessing the inhibition by bacteria of colony growth on agar media, (2) the inhibition of colony growth from colonized hemp seeds in liquid media and (3) the inhibition of cyst germination in liquid media. Finally, the fungicidal effect of the 24 most inhibitory bacterial species, and the inhibitory activity of their culture supernatants, was tested in the same way. Isolates identified as Aeromonas piscicola, A. sobria, Pantoea agglomerans and Pseudomonas fluorescens achieved the highest inhibition against S. parasitica. Many of these inhibitory isolates were obtained primarily from skin lesions of fish with saprolegniosis. It is suggested that some of these isolates might be useful in the biological control of saprolegniosis.     

Observations on saprolegniasis of adult sockeye salmon, Oncorhynchus nerka (Walbaum)

Some observations on the mycology and pathology of saprolegniasis of maturing sockeye salmon are reported. Saprolegnia spp. were isolated from all lesions examined. Some isolates did not produce oogonia and could not be identified further. The other isolates have affinities to the S. diclina-S. parasitica complex. Observations on the gross and histo-pathology of the lesions show the fungi can be active pathogens. It is hypothesized that increased levels of plasma corticosteroids and depletion of ascorbic acid reserves in the fish increase the probability that Saprolegnia spp. will initiate infections, either alone or concurrently with other opportunistic parasites.     

A Saprolegnia parasitica challenge system for rainbow trout: assessment of Pyceze as an anti-fungal agent for both fish and ova.

A reproducible Saprolegnia parasitica spore delivery system was developed and demonstrated to be effective in providing a sustained spore challenge for up to 10 d. Treatment of rainbow trout with slow-release intraperitoneal implants containing cortisol resulted in chronically elevated blood cortisol levels and rendered the fish susceptible to infection by S. parasitica when exposed to the spore challenge. Sham-implanted fish were not susceptible to infection. Bronopol (2-bromo-2-nitro-propane-1,3-diol), formulated as Pyceze, was effective in protecting predisposed fish from infection by S. parasitica when administered as a daily bath/flush treatment at concentrations of 15 mg l-1 and greater. Pyceze was also demonstrated to protect fertilised rainbow trout ova from S. parasitica challenge when administered as a daily bath/flush treatment at concentrations of between 30 and 100 mg l-1. Pyceze appears to qualify as a safe and effective replacement for malachite green and formalin in the prevention of fungal infections in the aquaculture environment.     

Functional characterization of the pleckstrin homology domain of a cellulose synthase from the oomycete Saprolegnia monoica

Some oomycetes, for instance Saprolegnia parasitica, are severe fish pathogens that cause important economic losses worldwide. Cellulose biosynthesis is a vital process for this class of microorganisms, but the corresponding molecular mechanisms are poorly understood. Of all cellulose synthesizing enzymes known, only some oomycete cellulose synthases contain a pleckstrin homology (PH) domain. Some human PH domains bind specifically to phosphoinositides, but most PH domains bind phospholipids in a non-specific manner. In addition, some PH domains interact with various proteins. Here we have investigated the function of the PH domain of cellulose synthase 2 from the oomycete Saprolegnia monoica (SmCesA2), a species closely related to S. parasitica. The SmCesA2 PH domain is similar to the C-terminal PH domain of the human protein TAPP1. It binds in vitro to phosphoinositides, F-actin and microtubules, and co-localizes with F-actin in vivo. Our results suggest a role of the SmCesA2 PH domain in the regulation, trafficking and/or targeting of the cell wall synthesizing enzyme.     

Insemination of the salmon spawn by Micromycetes at the fish-rearing farm "Pushcha-Voditsa"

Fungi of 21 species attributed to 11 genera of three classes--oomycetes, zygomycetes, deuteromycetes--have been isolated from the surface of the Salmo gairdneri Rich. spawn and from water samples of incubation apparatuses of the fish-rearing farm "Pushcha-Voditsa" (the Kiev Region). Among the revealed micromycetes only Saprolegnia parasitica C o k e r is a fish parasite. Most of isolates are imperfect fungi; they dominate among the hyphal forms in the mycobiota of fish and water and in natural water bodies. Fusarium sporotrichiella. B i l a i, one of the isolates, is slightly toxicogenic. It affects the fish spawn viability and biochemical indices: the joint incubation of spawn and fungi induces changes in the level of water-soluble protein, amylolytic and phosphatase activity. The disturbance of the spawn envelope integrity has been observed in the experiment.     

Functional characterization of a tyrosinase gene from the oomycete Saprolegnia parasitica by RNAi silencing

Here we describe the first application of transient gene silencing in Saprolegnia parasitica, a pathogenic oomycete that infects a wide range of fish, amphibians, and crustaceans. A gene encoding a putative tyrosinase from S. parasitica, SpTyr, was selected to investigate the suitability of RNA-interference (RNAi) to functionally characterize genes of this economically important pathogen. Tyrosinase is a mono-oxygenase enzyme that catalyses the O-hydroxylation of monophenols and subsequent oxidation of O-diphenols to quinines. These enzymes are widely distributed in nature, and are involved in the melanin biosynthesis. Gene silencing was obtained by delivering in vitro synthesized SpTyr dsRNA into protoplasts. Expression analysis, tyrosinase activity measurements, and melanin content analysis confirmed silencing in individual lines. Silencing of SpTyr resulted in a decrease of tyrosinase activity between 38 % and 60 %, dependent on the level of SpTyr-expression achieved. The SpTyr-silenced lines displayed less pigmentation in developing sporangia and occasionally an altered morphology. Moreover, developing sporangia from individual silenced lines possessed a less electron dense cell wall when compared to control lines, treated with GFP-dsRNA. In conclusion, the tyrosinase gene of S. parasitica is required for melanin formation and transient gene silencing can be used to functionally characterize genes in S. parasitica.     

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.     

Saprolegniosis in salmonids and their eggs in Japan

An epizootic of the fungal infection saprolegniosis that occurred in freshwater-cultured salmons and their eggs at some hatcheries in Hokkaido (Japan) was investigated. In almost all cases, the initial clinical sign was characterized by the growth of cotton-like mycelia on the fishs' body surface, especially the head, adipose fin, and caudal fin, but the mycelia were not visible to the naked eye in the internal organs. Thirty-three strains isolated from lesions were classified in the genus Saprolegnia according to their morphological and biological characteristics on hemp seed cultures at various temperatures. Fifteen of the strains were identified as Saprolegnia parasitica, 16 were identified as S. salmonis, and two were identified as S. australis.     

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.     

Mass mortality in cultured coho salmon (Oncorhynchus kisutch) due to Saprolegnia parasitica coker.

Epizootics of saprolegniasis occurred in 20 to 60 g freshwater-cultured coho salmon (Oncorhynchus kisutch) in Miyagi Prefecture, Japan. Cotton-like mycelia occurred on the body surface of infected fish, especially around the head, the adipose fin and the caudal fin, and aseptate hyphae occurred in the lesions. The hyphae also penetrated into the muscle and blood vessels. The isolated fungus was identified by asexual morphological characteristics as Saprolegnia parasitica (syn. S. diclina Type 1), a known salmonid fish pathogen.     

Distinctive Expansion of Potential Virulence Genes in the Genome of the Oomycete Fish Pathogen Saprolegnia parasitica

Oomycetes in the class Saprolegniomycetidae of the Eukaryotic kingdom Stramenopila have evolved as severe pathogens of amphibians, crustaceans, fish and insects, resulting in major losses in aquaculture and damage to aquatic ecosystems. We have sequenced the 63 Mb genome of the fresh water fish pathogen, Saprolegnia parasitica. Approximately 1/3 of the assembled genome exhibits loss of heterozygosity, indicating an efficient mechanism for revealing new variation. Comparison of S. parasitica with plant pathogenic oomycetes suggests that during evolution the host cellular environment has driven distinct patterns of gene expansion and loss in the genomes of plant and animal pathogens. S. parasitica possesses one of the largest repertoires of proteases (270) among eukaryotes that are deployed in waves at different points during infection as determined from RNA-Seq data. In contrast, despite being capable of living saprotrophically, parasitism has led to loss of inorganic nitrogen and sulfur assimilation pathways, strikingly similar to losses in obligate plant pathogenic oomycetes and fungi. The large gene families that are hallmarks of plant pathogenic oomycetes such as Phytophthora appear to be lacking in S. parasitica, including those encoding RXLR effectors, Crinkler''s, and Necrosis Inducing-Like Proteins (NLP). S. parasitica also has a very large kinome of 543 kinases, 10% of which is induced upon infection. Moreover, S. parasitica encodes several genes typical of animals or animal-pathogens and lacking from other oomycetes, including disintegrins and galactose-binding lectins, whose expression and evolutionary origins implicate horizontal gene transfer in the evolution of animal pathogenesis in S. parasitica.     

Properties of the soluble arachidonic acid 15-lipoxygenase and 15-hydroperoxide isomerase from the oomycete Saprolegnia parasitica

The soluble hydroperoxide isomerase and 15-lipoxygenase activities were partially purified from the oomycete Saprolegnia parasitica and some of their properties characterized. Both enzymes co-eluted with a molecular weight of 145,000-150,000 on Sephacryl S-300 chromatography. The enzyme activities also co-eluted on DEAE Sephadex ion exchange chromatography and hydroxylapatite chromatography. Both activities showed similar responses to pH and temperature. Both enzymes showed parallel inhibition by p-hydroxymercuribenzoate and eicosatetraynoic acid. The partially purified hydroperoxide isomerase showed an apparent km of 166 microM and a Vmax of 5.3 mumol/min/mg protein for exogenous 15-HPETE. It was not stimulated by calcium. These results suggest that the soluble hydroperoxide isomerase and 15-lipoxygenase activities from S. parasitica are both contained on the same protein or protein complex.     

Ecosystem screening approach for pathogen-associated microorganisms affecting host disease

The microbial community in which a pathogen evolves is fundamental to disease outcome. Species interacting with a pathogen on the host surface shape the distribution, density, and genetic diversity of the inoculum, but the role of these species is rarely determined. The screening method developed here can be used to characterize pathogen-associated species affecting disease. This strategy involves three steps: (i) constitution of the microbial community, using the pathogen as a trap; (ii) community selection, using extracts from the pathogen as the sole nutrient source; and (iii) molecular identification and the screening of isolates focusing on their effects on the growth of the pathogen in vitro and host disease. This approach was applied to a soilborne plant pathogen, Phytophthora parasitica, structured in a biofilm, for screening the microbial community from the rhizosphere of Nicotiana tabacum (the host). Two of the characterized eukaryotes interfered with the oomycete cycle and may affect the host disease. A Vorticella species acted through a mutualistic interaction with P. parasitica, disseminating pathogenic material by leaving the biofilm. A Phoma species established an amensal interaction with P. parasitica, strongly suppressing disease by inhibiting P. parasitica germination. This screening method is appropriate for all nonobligate pathogens. It allows the definition of microbial species as promoters or suppressors of a disease for a given biotope. It should also help to identify important microbial relationships for ecology and evolution of pathogens.     

Saprolegniaceae identified on amphibian eggs throughout the Pacific Northwest, USA, by internal transcribed spacer sequences and phylogenetic analysis

We assessed the diversity and phylogeny of Saprolegniaceae on amphibian eggs from the Pacific Northwest, with particular focus on Saprolegnia ferax, a species implicated in high egg mortality. We identified isolates from eggs of six amphibians with the internal transcribed spacer (ITS) and 5.8S gene regions and BLAST of the GenBank database. We identified 68 sequences as Saprolegniaceae and 43 sequences as true fungi from at least nine genera. Our phylogenetic analysis of the Saprolegniaceae included isolates within the genera Saprolegnia, Achlya and Leptolegnia. Our phylogeny grouped S. semihypogyna with Achlya rather than with the Saprolegnia reference sequences. We found only one isolate that grouped closely with S. ferax, and this came from a hatchery-raised salmon (Idaho) that we sampled opportunistically. We had representatives of 7-12 species and three genera of Saprolegniaceae on our amphibian eggs. Further work on the ecological roles of different species of Saprolegniaceae is needed to clarify their potential importance in amphibian egg mortality and potential links to population declines.     

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.     

鳙团移核鱼LDH,MDH同工酶的研究

对二龄鳙鱼细胞核和团头鲂细胞质配合的核质杂种鱼－－鳙团移核鱼及其亲本,供核体鳙鱼和受核体团头鲂肌组织ＬＤＨ、ＭＤＨ同工酶进行了研究试验。鳙团移核鱼和供核体鳙鱼肌组织ＬＤＨ同工酶均具有ＬｄｈＡ２Ｂ２一条谱带;受核体团头鲂的则具有ＬｄｈＡ２、ＬｄｈＡ２Ｂ１、ＬｄｈＡ２Ｂ２、ＬｄｈＡ１Ｂ３、ＬｄｈＢ４等五条谱带。移核鱼和供核体鳙鱼肌组织的ＭＤＨ同工酶都各具有二条谱带：Ｓ－ｍｄｈＡ２、Ｓ－ｍｄｈＡＢ;受核     

德国小蠊生活史各期蛋白质、糖与酯酶同功酶的比较研究

德国小蠊生活史各期经等电聚焦后,用考马斯亮兰染蛋白质,然后用薄层层析扫描仪扫描,卵、若虫、成虫分别显带36、30和43条;用PAS染糖,分别显带29、21和33条。各阶段所显区带由糖蛋白、蛋白质和多糖组成。另补,本还对各阶段的酯酶同功酶进行了比较．用坚牢兰染色后．卵、若虫、成虫分别显带23、23和25条。结果表明．每种物质均以成虫期显带较多。