Use of Onion (Allium cepa) and Garlic (Allium sativum) Wastes for the Prevention of Fungal Disease (Saprolegnia parasitica) on Eggs of Rainbow Trout (Oncorhynchus mykiss).

This study was designed to prevent fungal disease (Saprolegnia parasitica) that occurs on rainbow trout eggs (Oncorhynchus mykiss) by using wastes of onion (Allium cepa) and garlic (Allium sativum) plants. For this purpose, fertilized rainbow trout eggs were exposed to garlic skin, garlic stem and onion skin aqueous methanolic extracts by bathing in concentrations of 0.4, 0.8, 1.6 and 3.2 g/L, whereas the control group was left untreated. The larvae in all groups were monitored until they become free-swimming larvae, the number of eggs died due to fungus was recorded, and the data obtained from experimental groups and control group were compared. As a result, it was determined that onion skin had no effect on the number of eggs that died due to fungus (p > .05), while garlic skin and garlic stem extracts significantly reduced the number of fungal infestations without affecting the number of live larvae or the embryological development of the eggs (p < .05). In light of these data, we conclude that garlic skin (0.4, 0.8, and 1.6 g/L) and garlic stem (0.8 and 1.6 g/L) aqueous methanolic extracts are effective at preventing Saprolegnia parasitica infestation on rainbow trout eggs and may be used in aquaculture.

Purinergic signaling creates an anti-inflammatory profile in spleens of grass carp Ctenopharyngodon idella naturally infected by Saprolegnia parasitica: An attempt to prevent ATP pro-inflammatory effects.

Extracellular adenosine triphosphate (ATP) is as key mediator of immune and inflammatory responses. ATP is normally sequestered in the intracellular milieu and released by apoptotic and necrotic cells, where it acts as a pro-inflammatory mediator in the extracellular milieu. A limited number of studies have explored the involvement of purinergic signaling in oomycete infections, including Saprolegnia parasitica; this is a most destructive oomycete pathogen, associated with high mortality and severe economic losses for fish producers. The aim of this study was to determine whether purinergic signaling exerts anti- or pro-inflammatory effects in spleens of grass carp (Ctenopharyngodon idella) naturally infected by S. parasitica. Animals naturally infected with S. parasitica showed typical gross lesions characterized by cotton-wool tufts on the tail and fins, as well as severe histopathological lesions such as necrosis. Spleen ATP and metabolites of nitric oxide (NOx) levels were higher in fish naturally infected by S. parasitica compared to control on day 7 post-infection (PI). Spleen nucleoside triphosphate diphosphohydrolase (NTPDase) activity (ATP as substrate) was greater in fish naturally infected by S. parasitica than in uninfected on day 7 PI, while no significant differences were observed between groups with respect to NTPDase (adenosine diphosphate as substrate) and 5'-nucleotidase activities. Finally, adenosine deaminase (ADA) activity was lower in fish naturally infected by S. parasitica than in uninfected fish on day 7 PI. In summary, spleen tissue necrosis in the context of saprolegniosis provokes an intense release of ATP into the extracellular milieu, where it interacts with the P2X7 purine receptor and leads to a self-sustained pro-inflammatory deleterious cycle, contributing to an intense inflammatory process. In response to excessive ATP levels in the extracellular milieu, ATP and adenosine hydrolysis were modulated in an attempt to restrict the inflammatory process via upregulation of NTPDase and downregulation of ADA activities. We conclude that the purinergic signaling pathway modulates immune and inflammatory responses during natural infection with S. parasitica.

Impact of short-term temperature challenges on the larvicidal activities of the entomopathogenic watermold Leptolegnia chapmanii against Aedes aegypti, and development on infected dead larvae.

The oomycete Leptolegnia chapmanii is among the most promising entomopathogens for biological control of Aedes aegypti. This mosquito vector breeds in small water collections, where this aquatic watermold pathogen can face short-term scenarios of challenging high or low temperatures during changing ambient conditions, but it is yet not well understood how extreme temperatures might affect the virulence and recycling capacities of this pathogen. We tested the effect of short-term exposure of encysted L. chapmanii zoospores (cysts) on A. aegypti larvae killed after infection by this pathogen to stressful low or high temperatures on virulence and production of cysts and oogonia, respectively. Cysts were exposed to temperature regimes between -12 °C and 40 °C for 4, 6 or 8 h, and then their infectivity was tested against third instar larvae (L3) at 25 °C; in addition, production of cysts and oogonia on L3 killed by infection exposed to the same temperature regimes as well as their larvicidal activity were monitored. Virulence of cysts to larvae and the degree of zoosporogenesis on dead larvae under laboratory conditions were highest at 25 °C but were hampered or even blocked after 4 up to 8 h exposure of cysts or dead larvae at both the highest (35 °C and 40 °C) and the lowest (-12 °C) temperatures followed by subsequent incubation at 25 °C. The virulence of cysts was less affected by accelerated than by slow thawing from the frozen state. The production of oogonia on dead larvae was stimulated by short-term exposure to freezing temperatures (-12 °C and 0 °C) or cool temperatures (5 °C and 10 °C) but was not detected at higher temperatures (25 °C-40 °C). These findings emphasize the susceptibility of L. chapmanii to short-term temperature stresses and underscore its interest as an agent for biocontrol of mosquitoes in the tropics and subtropics, especially A. aegypti, that breed preferentially in small volumes of water that are generally protected from direct sunlight.

Galleria melonella as an experimental in vivo host model for the fish-pathogenic oomycete Saprolegnia parasitica.

Oomycetes are eukaryotic pathogens infecting animals and plants. Amongst them Saprolegnia parasitica is a fish pathogenic oomycete causing devastating losses in the aquaculture industry. To secure fish supply, new drugs are in high demand and since fish experiments are time consuming, expensive and involve animal welfare issues the search for adequate model systems is essential. Galleria mellonella serves as a heterologous host model for bacterial and fungal infections. This study extends the use of G. mellonella for studying infections with oomycetes. Saprolegniales are highly pathogenic to the insects while in contrast, the plant pathogen Phytophthora infestans showed no pathogenicity. Melanisation of hyphae below the cuticle allowed direct macroscopic monitoring of disease progression. However, the melanin response is not systemic as for other pathogens but instead is very local. The mortality of the larvae is dose-dependent and can be induced by cysts or regenerating protoplasts as an alternative source of inoculation.

Specialized attachment structure of the fish pathogenic oomycete Saprolegnia parasitica.

The secondary cysts of the fish pathogen oomycete Saprolegnia parasitica possess bundles of long hooked hairs that are characteristic to this economically important pathogenic species. Few studies have been carried out on elucidating their specific role in the S. parasitica life cycle and the role they may have in the infection process. We show here their function by employing several strategies that focus on descriptive, developmental and predictive approaches. The strength of attachment of the secondary cysts of this pathogen was compared to other closely related species where bundles of long hooked hairs are absent. We found that the attachment of the S. parasitica cysts was around three times stronger than that of other species. The time sequence and influence of selected factors on morphology and the number of the bundles of long hooked hairs conducted by scanning electron microscopy study revealed that these are dynamic structures. They are deployed early after encystment, i.e., within 30 sec of zoospore encystment, and the length, but not the number, of the bundles steadily increased over the encystment period. We also observed that the number and length of the bundles was influenced by the type of substrate and encystment treatment applied, suggesting that these structures can adapt to different substrates (glass or fish scales) and can be modulated by different signals (i.e., protein media, 50 mM CaCl2 concentrations, carbon particles). Immunolocalization studies evidenced the presence of an adhesive extracellular matrix. The bioinformatic analyses of the S. parasitica secreted proteins showed that there is a high expression of genes encoding domains of putative proteins related to the attachment process and cell adhesion (fibronectin and thrombospondin) coinciding with the deployment stage of the bundles of long hooked hairs formation. This suggests that the bundles are structures that might contribute to the adhesion of the cysts to the host because they are composed of these adhesive proteins and/or by increasing the surface of attachment of this extracellular matrix.

Hemi-Synthesis and Anti-Oomycete Activity of Analogues of Isocordoin.

An efficient synthesis of a series of 4'-oxyalkyl-isocordoin analogues (2-8) is reported for the first time. Their structures were confirmed by ¹H-NMR, 13C-NMR, and HRMS. Their anti-oomycete activity was evaluated by mycelium and spores inhibition assay against two selected pathogenic oomycetes strains: Saprolegnia parasitica and Saprolegnia australis. The entire series of isocordoin derivatives (except compound 7) showed high inhibitory activity against these oomycete strains. Among them, compound 2 exhibited strong activity, with minimum inhibitory concentration (MIC) and minimum oomyceticidal concentration (MOC) values of 50 µg/mL and 75 µg/mL, respectively. The results showed that 4'-oxyalkylated analogues of isocordoin could be potential anti-oomycete agents.

Comparative analysis of sterol acquisition in the oomycetes Saprolegnia parasitica and Phytophthora infestans.

The oomycete class includes pathogens of animals and plants which are responsible for some of the most significant global losses in agriculture and aquaculture. There is a need to replace traditional chemical means of controlling oomycete growth with more targeted approaches, and the inhibition of sterol synthesis is one promising area. To better direct these efforts, we have studied sterol acquisition in two model organisms: the sterol-autotrophic Saprolegnia parasitica, and the sterol-heterotrophic Phytophthora infestans. We first present a comprehensive reconstruction of a likely sterol synthesis pathway for S. parasitica, causative agent of the disease saprolegniasis in fish. This pathway shows multiple potential routes of sterol synthesis, and draws on several avenues of new evidence: bioinformatic mining for genes with sterol-related functions, expression analysis of these genes, and analysis of the sterol profiles in mycelium grown in different media. Additionally, we explore the extent to which P. infestans, which causes the late blight in potato, can modify exogenously provided sterols. We consider whether the two very different approaches to sterol acquisition taken by these pathogens represent any specific survival advantages or potential drug targets.

Elucidating the Diversity of Aquatic Microdochium and Trichoderma Species and Their Activity against the Fish Pathogen Saprolegnia diclina.

Animals and plants are increasingly threatened by emerging fungal and oomycete diseases. Amongst oomycetes, Saprolegnia species cause population declines in aquatic animals, especially fish and amphibians, resulting in significant perturbation in biodiversity, ecological balance and food security. Due to the prohibition of several chemical control agents, novel sustainable measures are required to control Saprolegnia infections in aquaculture. Previously, fungal community analysis by terminal restriction fragment length polymorphism (T-RFLP) revealed that the Ascomycota, specifically the genus Microdochium, was an abundant fungal phylum associated with salmon eggs from a commercial fish farm. Here, phylogenetic analyses showed that most fungal isolates obtained from salmon eggs were closely related to Microdochium lycopodinum/Microdochium phragmitis and Trichoderma viride species. Phylogenetic and quantitative PCR analyses showed both a quantitative and qualitative difference in Trichoderma population between diseased and healthy salmon eggs, which was not the case for the Microdochium population. In vitro antagonistic activity of the fungi against Saprolegnia diclina was isolate-dependent; for most Trichoderma isolates, the typical mycoparasitic coiling around and/or formation of papilla-like structures on S. diclina hyphae were observed. These results suggest that among the fungal community associated with salmon eggs, Trichoderma species may play a role in Saprolegnia suppression in aquaculture.

Saprolegnia diclina IIIA and S. parasitica employ different infection strategies when colonizing eggs of Atlantic salmon, Salmo salar L.

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).

Effect of ultraviolet-A radiation on the production of Leptolegnia chapmanii (Saprolegniales: Saprolegniaceae) zoospores on dead Aedes aegypti (Diptera: Culicidae) larvae and their larvicidal activity.

Impact of UV-radiation in entomopathogens in aquatic environments remains little investigated. The present study reports on the effect of UV-A on the larvicidal activity of Leptolegnia chapmanii zoospores in Aedes aegypti; on the production of zoospores in larvae killed by the pathogen and then exposed to UV-A; and on the activity of these zoospores against healthy larvae. Whereas the virulence of free zoospores in A. aegypti larvae was affected by a UV-A exposure time longer than 10min, production of zoospores in larvae and their virulence were not hampered at a maximal 8h exposure of dead larvae to UV-A. Findings suggest that dead larvae and zoosporangia provide a certain protection to zoospores against UV-A and emphasize the susceptibility of free encysted zoospores to such radiation.

In vitro passages impact on virulence of Saprolegnia parasitica to Atlantic salmon, Salmo salar L. parr.

The effect of serial in vitro subculturing on three pathogenic strains of Saprolegnia parasitica was investigated. The isolates were passed through Atlantic salmon, Salmo salar L. parr, and then re-isolated as single spore colonies. All strains caused infection. The isolate obtained from diseased fish served as a virulent reference culture and was designated 'AP' ('activated through passage'). Successive subculturing was made by obtaining an inoculum from AP to produce the 2nd subculture and then passaged to the 3rd subculture (from the 2nd), until the 15th passage was obtained. Spores used to produce storage cultures were collected at passages 5, 10 and 15. The different passages of each strain were used to artificially infect Atlantic salmon parr. Morphological characterization of growth patterns was performed to observe differences occurring due to serial in vitro subculturing. Two of the strains declined in virulence after 15 successive in vitro subcultures, whereas one did not. This study is the first to investigate attenuation of virulence in Saprolegnia and whether or not isolates of S. parasitica should be passed through the fish host prior to challenge experiments. It reveals that some strains degenerate more rapidly than others when subjected to successive in vitro subculturing on glucose-yeast extract.

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.

Do fathead minnows, Pimephales promelas Rafinesque, alter their club cell investment in responses to variable risk of infection from Saprolegnia?

Fish in the Superorder Ostariophysi possess large epidermal club cells that release chemical cues warning nearby conspecifics of danger. Despite the long-held assumption that such club cells evolved under the selective force of predation, recent studies demonstrated that predation has no effect on club cell investment. Rather, club cells have an immune function and cell production may be stimulated by skin-penetrating pathogens and parasites. The current work investigates whether fathead minnows, Pimephales promelas, alter their club cell characteristics based on variation in infection risk. In a 2 × 3 design, we exposed minnows to infective cysts of two oomycete species (Saprolegnia ferax and S. parasitica) at three different concentrations (2, 20 or 200 cysts L(-1)). Club cell characteristics (number and size) were quantified 12 days after exposure. Saprolegnia parasitica is thought to be more pathogenic than S. ferax, hence we predicted greater club cell investment and a larger turnover rate of cells by minnows exposed to S. parasitica than S. ferax. We also predicted that minnows exposed to higher numbers of cysts should invest more in club cells and have a higher turnover rate of cells. We found no difference in club cell density or size between fish exposed to the two Saprolegnia species; however, fish exposed to high concentrations of pathogens had smaller club cells than those exposed to low concentrations, indicating a higher rate of turnover of cells in the epidermis.

Pathogenicity of Saprolegnia spp. to Atlantic salmon, Salmo salar L., eggs.

Live and dead Atlantic salmon eyed eggs were challenged with eight different Saprolegnia isolates, selected because of their varied origins, known morphological characteristics and growth/germination pattern. Some isolates were also tested for pathogenicity to Atlantic salmon parr. Challenge of eggs was performed by exposure to spores in suspension or by co-incubation of live eggs with infected dead eggs. The phenotypic characteristics of the isolates were evaluated in relation to their observed pathogenicity from the challenge experiment, to identify possible virulence factors leading to egg-infection by Saprolegnia. The results from the experiments confirm that live eggs are refractory to infection with Saprolegnia spores in suspension and that an infection of live eggs can only occur from an infection nucleus represented by dead eggs or debris. It was observed that strains pathogenic to salmon parr were not particularly infective towards eggs, and the isolates that gave the highest infection rates to eggs were species considered to be saprotrophs.

Differences in susceptibility to Saprolegnia infections among embryonic stages of two anuran species.

Many amphibians are known to suffer embryonic die-offs as a consequence of Saprolegnia infections; however, little is known about the action mechanisms of Saprolegnia and the host-pathogen relationships. In this study, we have isolated and characterized the species of Saprolegnia responsible for infections of embryos of natterjack toad (Bufo calamita) and Western spadefoot toad (Pelobates cultripes) in mountainous areas of Central Spain. We also assessed the influence of the developmental stage within the embryonic period on the susceptibility to the Saprolegnia species identified. Only one strain of Saprolegnia was isolated from B. calamita and identified as S. diclina. For P. cultripes, both S. diclina and S. ferax were identified. Healthy embryos of both amphibian species suffered increased mortality rates when exposed to the Saprolegnia strains isolated from individuals of the same population. Embryonic developmental stage was crucial in determining the sensitivity of embryos to Saprolegnia infection. The mortalities of P. cultripes and B. calamita embryos exposed at Gosner stages 15 (rotation) and 19 (heart beating) were almost total 72 h after challenge with Saprolegnia, while those exposed at stage 12 (late gastrula) showed no significant effects at that time. This is the first study to demonstrate the role of embryonic development on the sensitivity of amphibians to Saprolegnia.

Further research on the production, longevity and infectivity of the zoospores of Leptolegnia chapmanii Seymour (Oomycota: Peronosporomycetes).

The effect of temperature on the production, survival and infectivity of zoospores of an Argentinean isolate of Leptolegnia chapmanii was determined under laboratory conditions. Production of zoospores of L. chapmaniiin vitro and in vivo upon first and fourth instars larvae of the mosquito Aedes aegypti was studied at three different temperatures. Zoospores from infected larvae were infective to mosquito larvae for 51, 12, and 5 consecutive days when maintained at 25, 35, and 10 degrees C, respectively. Maximum zoospore production in infected fourth-instar larvae was 9.6+/-1.4x10(4) zoosp/larva at 48 h at 25 degrees C. The average number of zoospores produced by individual fourth-instar Ae. aegypti larvae infected with L. chapmanii was 3.57+/-0.46x10(5) zoospores during 6 consecutive days at 25 degrees C. Zoospore production in vitro was also affected by temperature with a maximum of zoospores (n=47,666/ml) produced at 25 degrees C. When zoospores produced in vitro were used as inoculum against Ae. aegypti larvae at 25 degrees C, larval mortality was recorded for 5 consecutive weeks. The encystment process for zoospores took 17-20 min; the germination of cysts (excystment) occurred 5 min after exposure in water to mosquito larvae. The minimal time of contact between zoospores and mosquito larvae to develop infection was two minutes. Infection took place by zoospore attachment onto and then penetration through the larval cuticle or by ingestion of cysts as was confirmed by histological studies. Temperature directly affected infectivity and production of zoospores in vivo and in vitro although L. chapmanii zoospores tolerate a wide range of temperatures.

Effects of a fertilizer, an insecticide, and a pathogenic fungus on hatching and survival of bullfrog (Rana catesbeiana) tadpoles.

We assessed the single and interactive effects of ammonium nitrate fertilizer, the insecticide carbaryl, and a widespread waterborne pathogen Saprolegnia ferax on the hatching and survival of bullfrog (Rana catesbeiana) tadpoles in the laboratory. Carbaryl significantly reduced tadpole survival and hatching rate, but no interactive effects were found among stressors.

Effects of temperature, pH and salinity on the infection of Leptolegnia chapmanii Seymour (Peronosporomycetes) in mosquito larvae.

The effects of temperature, pH, and NaCl concentrations on the infectivity of zoospores of Leptolegnia chapmanii (Argentine isolate) were determined for Aedes aegypti and Culex pipiens under laboratory conditions. Zoospores of L. chapmanii were infectious at temperatures between 10 and 35 degrees C but not at 5 or 40 degrees C. At the permissive temperatures, mortality rates in young instars were much higher than in older instars and larvae of Ae. aegypti were more susceptible to L. chapmanii than larvae of Cx. pipiens. At 25 degrees C, Ae. aegypti larvae challenged with L. chapmanii zoospores resulted in 100% infection at pH levels ranging from 4 to 10. Larvae of Cx. pipiens exposed to similar pH and zoospore concentrations resulted in increasing mortality rates from 62% to 99% at pH 4 to 7, respectively, and then decreased to 71% at pH 10. Aedes aegypti larvae exposed to L. chapmanii zoospores in NaCl concentrations ranging from 0 to 7 parts per thousand (ppt) at 25 degrees C resulted in 100% mortality while mortality rates for Cx. pipiens decreases from 96% in distilled water to 31.5% in water with 6 ppt NaCl. Control Cx. pipiens larvae died when exposed at a NaCl concentration of 7 ppt. Vegetative growth of L. chapmanii was negatively affected by NaCl concentrations. These results have demonstrated that the Argentinean isolate of L. chapmanii tolerated a wide range of temperatures, pH, and salinity, suggesting that it has the potential to adapt to a wide variety of mosquito habitats.

Effects of nitrate and the pathogenic water mold Saprolegnia on survival of amphibian larvae.

We tested for a synergism between nitrate and Saprolegnia, a pathogenic water mold, using larvae of 3 amphibian species: Ambystoma gracile (northwestern salamander), Hyla regilla (Pacific treefrog) and Rana aurora (red-legged frog). Each species was tested separately, using a 3 x 2 fully factorial experiment with 3 nitrate treatments (none, low and high) and 2 Saprolegnia treatments (Saprolegnia and control). Survival of H. regilla was not affected significantly by either experimental factor. In contrast, survival of R. aurora was affected by a less-than-additive interaction between Saprolegnia and nitrate. Survival of R. aurora was significantly lower in the Saprolegnia compared to the control treatment when nitrate was not added, but there was no significant difference in survival between Saprolegnia and control treatments in the low and high nitrate treatments, consistent with increased nitrate preventing Saprolegnia from causing mortality of R. aurora. Survival of A. gracile followed a similar pattern, but the difference between Saprolegnia and control treatments when nitrate was not added was not significant, nor was the nitrate x Saprolegnia interaction. Our study suggests that Saprolegnia can cause mortality in amphibian larvae, that there are interspecific differences in susceptibility and that the effects of Saprolegnia on amphibians are context-dependent.

Morphological and physiological characteristics of Saprolegnia spp. strains pathogenic to Atlantic salmon, Salmo salar L.

Seventeen strains of Saprolegnia spp. were examined for morphological and physiological characteristics, and seven were examined for their pathogenicity to Atlantic salmon, Salmo salar L. Two of the Saprolegnia strains tested caused 89 and 31% cumulative mortality in challenged salmonids and were significantly more pathogenic than the other strains tested. The positive control (Saprolegnia parasitica ATCC 90213) caused 18% mortality, but this was not significantly higher than non-pathogenic strains (0-3% cumulative mortality). All the pathogenic Saprolegnia strains and two non-pathogenic strains had secondary cysts with long, hooked hairs, a characteristic which is claimed to be typical of S. parasitica. This characteristic is apparently necessary, but does not in itself determine the ability to cause mortality in Atlantic salmon. However, all the pathogenic Saprolegnia strains in the present study showed a significantly higher initial growth rate of cysts in sterilized tap water than did non-pathogenic strains. The results of the present study suggest that initial growth rate of germinating cysts in pure water, together with the presence of long hooked hairs on the secondary cysts, may be indicators of pathogenicity of Saprolegnia strains to Atlantic salmon.