Saprolegnia parasitica is a freshwater oomycete that is capable of infecting several species of fin fish. Saprolegniosis, the disease caused by this microbe, has a substantial impact on Atlantic ...salmon aquaculture. No sustainable treatment against saprolegniosis is available, and little is known regarding the host response. In this study, we examined the immune response of Atlantic salmon to S. parasitica infection and to its cell wall carbohydrates. Saprolegnia triggers a strong inflammatory response in its host (i.e., induction of interleukin-1β1 IL-1β1, IL-6, and tumor necrosis factor alpha), while severely suppressing the expression of genes associated with adaptive immunity in fish, through downregulation of T-helper cell cytokines, antigen presentation machinery, and immunoglobulins. Oomycete cell wall carbohydrates were recognized by fish leukocytes, triggering upregulation of genes involved in the inflammatory response, similar to what is observed during infection. Our data suggest that S. parasitica is capable of producing prostaglandin corrected E2 (PGE2) in vitro, a metabolite not previously shown to be produced by oomycetes, and two proteins with homology to vertebrate enzymes known to play a role in prostaglandin biosynthesis have been identified in the oomycete genome. Exogenous PGE2 was shown to increase the inflammatory response in fish leukocytes incubated with cell wall carbohydrates while suppressing genes involved in cellular immunity (gamma interferon IFN-γ and the IFN-γ-inducible protein γ-IP). Inhibition of S. parasitica zoospore germination and mycelial growth by two cyclooxygenase inhibitors (aspirin and indomethacin) also suggests that prostaglandins may be involved in oomycete development.
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.
The mucosal surfaces of wild and farmed aquatic vertebrates face the threat of many aquatic pathogens, including fungi. These surfaces are colonized by diverse symbiotic bacterial communities that ...may contribute to fight infection. Whereas the gut microbiome of teleosts has been extensively studied using pyrosequencing, this tool has rarely been employed to study the compositions of the bacterial communities present on other teleost mucosal surfaces. Here we provide a topographical map of the mucosal microbiome of an aquatic vertebrate, the rainbow trout (Oncorhynchus mykiss). Using 16S rRNA pyrosequencing, we revealed novel bacterial diversity at each of the five body sites sampled and showed that body site is a strong predictor of community composition. The skin exhibited the highest diversity, followed by the olfactory organ, gills, and gut. Flectobacillus was highly represented within skin and gill communities. Principal coordinate analysis and plots revealed clustering of external sites apart from internal sites. A highly diverse community was present within the epithelium, as demonstrated by confocal microscopy and pyrosequencing. Using in vitro assays, we demonstrated that two Arthrobacter sp. skin isolates, a Psychrobacter sp. strain, and a combined skin aerobic bacterial sample inhibit the growth of Saprolegnia australis and Mucor hiemalis, two important aquatic fungal pathogens. These results underscore the importance of symbiotic bacterial communities of fish and their potential role for the control of aquatic fungal diseases.
Here, we address the morphological changes of eyed eggs of Atlantic salmon, Salmo salar L. infected with Saprolegnia from a commercial hatchery and after experimental infection. Eyed eggs infected ...with Saprolegnia spp. from 10 Atlantic salmon females were obtained. Egg pathology was investigated by light and scanning electron microscopy. Eggs from six of ten females were infected with S. parasitica, and two females had infections with S. diclina clade IIIA; two Saprolegnia isolates remained unidentified. Light microscopy showed S. diclina infection resulted in the chorion in some areas being completely destroyed, whereas eggs infected with S. parasitica had an apparently intact chorion with hyphae growing within or beneath the chorion. The same contrasting pathology was found in experimentally infected eggs. Scanning electron microscopy revealed that S. parasitica grew on the egg surface and hyphae were found penetrating the chorion of the egg, and re‐emerging on the surface away from the infection site. The two Saprolegnia species employ different infection strategies when colonizing salmon eggs. Saprolegnia diclina infection results in chorion destruction, while S. parasitica penetrates intact chorion. We discuss the possibility these infection mechanisms representing a necrotrophic (S. diclina) vs. a facultative biotrophic strategy (S. parasitica).
A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It ...was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (Ks, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (Kd, ≤3 nM) and prothioconazole-desthio the lowest (Kd, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC50s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC100, >256 μg ml(-1)). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC100 the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C, 16 to 64 μg ml(-1)) with the exception of clotrimazole, which was as potent as malachite green (MIC100, ∼1 μg ml(-1)). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, ∼1 μM) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC100, ∼1 to 2 μg ml(-1)) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.
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.
•Saprolegnia spp. are able to infect aquatic insects eg. stoneflies, caddisflies.•Saprolegnia spp. from insects can infect salmon eggs and frog embryos.•Pathogens of salmon eggs and frog embryos are also able to infect insects.•Saprolegnia isolates can infect/colonize multiple hosts.•Our findings are important for disease management of the affected hosts.
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
°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.
The present work is the first comprehensive study of fungus‐like stramenopilous organisms (Oomycota) diversity in Lithuanian fish farms aimed at proper identification of saprolegniasis pathogens, ...which is important for water quality control, monitoring infection levels and choosing more effective treatments for this disease in aquaculture. Pathogenic to fish, Saprolegnia and other potentially pathogenic water moulds were isolated from adult fish, their eggs, fry and from water samples. All detected isolates were examined morphologically and confirmed by sequence‐based molecular methods. A total of eight species belonging to the genera Saprolegnia, Achlya, Newbya and Pythium were identified. Four species (S. parasitica, S. ferax, S. australis and S. diclina) were found to be the main causative agents of saprolegniasis in Lithuania. S. parasitica and S. ferax dominated both in hatcheries and open fishponds, accounting for 66.2% of all isolates. S. parasitica was isolated from all farmed salmonid fish species as well as from the skin of Cyprinus carpio, Carassius carassius and Perca fluviatilis. S. australis was isolated from water and once from the skin of Oncorhynchus mykiss, and S. diclina was detected only once on the skin of Salmo salar fish. In addition, Achlya ambisexualis, Saprolegnia anisospora and Newbia oligocantha isolated during this study are noted as a possible source of saprolegniasis. The results of this study are relevant for assessing the risk of potential outbreaks of saprolegniasis or other saprolegnia‐like infection in Lithuanian freshwater aquaculture.
The rapid increase in the aquaculture production of salmonids has been followed by a rise in several diseases. In particular, saprolegniosis can account for at least 10% of the annual economic loss ...in salmonids. In this study, we investigated the main Saprolegnia species involved in saprolegniosis of salmonids in Chile, and their association with specific developmental stages of the host fish. For this purpose, we studied 244 isolates of Saprolegnia-affected Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and king salmon (Oncorhynchus tshawytscha) from the salmon farming regions, using a recently developed identification strategy based on molecular taxonomical operational units. We found that the Saprolegnia species associated with diseased salmon were Saprolegnia australis, Saprolegnia delica, Saprolegnia diclina, Saprolegnia ferax, Saprolegnia parasitica and two new Saprolegnia species observed during this study. In order to determine whether there were any specific species associations with different stages in the fish life cycle, we applied mosaic plots and correspondence analyses for categorical data. These analyses showed a strong association of S. parasitica with samples from the adult stage of the fish (χ2=196.29, p<0.0001), while the species S. australis, S. diclina and Saprolegnia sp. 2 were strongly associated with embryonic stages (eggs or alevins) (χ2=196.29, p<0.0001). This work represents the first detailed molecular characterization of Saprolegnia species involved in saprolegniosis in Chile, and the first study showing specific association of different Saprolegnia species with different stages in the salmonid life cycle.
•Infected salmonids from Chile can have up to seven Saprolegnia spp. associated with them.•Saprolegnia parasitica is associated with adult salmonids.•Saprolegnia australis, S. diclina and a Saprolegnia sp. are associated with embryonic stages.
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