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.
The description, identification and classification of organisms are the pillar in biodiversity and evolutionary studies. The fungal-like organism Saprolegnia contains important animal pathogens. ...However, its taxonomy is weak, making it difficult to perform further studies. This problem mainly arises from the unavailability of suitable holotypes. We propose a standardized protocol for describing Saprolegnia spp. that includes good cultural practices and proper holotype preservation. In order to illustrate this new proposal, we describe two species, Saprolegnia aenigmatica sp. nov. and Saprolegnia racemosa sp. nov., based on the recently described molecular operational taxonomic units (MOTUs), phylogenetic relationships, and the analyses of morphological features. We show that they belong to two different MOTUs that are grouped into two sister clades. Morphologically, we find that S. racemosa exhibits a species-specific character, i.e., aggrupation of oogonia in racemes, while S. aenigmatica does not have any specific characters. Analyses of a combined set of characters, i.e., length and breadth of sporangia, length/breadth ratio (l/b) of oogonia, cyst and oospore diameter, and the number of oospores per oogomium, allow distinguishing these two species. To improve Saprolegnia taxonomy, we propose to incorporate into the protologue: (i) several isolates of the new species; (ii) the rDNA sequences to compare them to data-bases of Saprolegnia sequences of reference; (iii) a phylogenetic analysis to check relationships with other species; (iv) to preserve holotypes in absolute ethanol and to include lyophilized material from holotype; and (v) the ex-type as a pure culture from single-spore isolates stored in at least two different collections.
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.
To identify the pathogens causing saprolegniosis among farmed fish in Nova Scotia, 172 infected tissues and 23 water samples were collected from six species of teleosts: Atlantic salmon (Salmo ...salar), brown trout (Salmo trutta), Arctic charr (Salvelinus alpinus), brook trout (Salvelinus fontinalis), striped bass (Morone saxatilis) and rainbow trout (Oncorhynchus mykiss) at nine facilities over a 600 km range. Following laboratory culture, 132 isolates were recovered. Six species of oomycetes were identified from analysis of the internal transcribed spacer (ITS) sequence of the nrDNA: Saprolegnia parasitica, Saprolegnia ferax, Saprolegnia diclina, Saprolegnia aenigmatica, Saprolegnia torulosa, Saprolegnia sp. and Pythiopsis cymosa. Further phylogenetic analyses of the ITS and cytochrome c oxidase subunit 1 (Cox1) regions revealed four strains of Saprolegnia parasitica (named here as S1, S2, S3 and S4), of which S1 and S2 were common (37% and 42% of the isolates), and two strains of S. ferax. Among S. parasitica, S2 and S3 are more closely related to each other than to S1 based on the phylogenetic analyses and predicted RNA secondary structure of the ITS region. Sexual structures with a similar morphology were formed by S1 and S3 in vitro, but were not formed by S2.
As one of the most serious pathogens in the freshwater aquatic environment, Saprolegnia can induce a high mortality rate during the fish egg incubation period. This study investigated the ...anti-Saprolegnia activity of a total of 108 plants on Saprolegnia parasitica in vitro and Dioscorea collettii was selected for further studies. By loading on an open silica gel column and eluting with petroleum ether–ethyl acetate–methanol, dioscin (C45H72O16) was isolated from D. collettii. Saprolegnia parasitica growth was inhibited significantly when dioscin concentration was more than 2.0 mg L−1. When compared with formalin and hydrogen peroxide, dioscin showed a higher inhibitory effect. As potential inhibition mechanisms, dioscin could cause the S. parasitica mycelium morphologic damage, dense folds, or disheveled protuberances observed by field emission scanning electron microscopy and the influx of Propidium iodide. The structural changes in the treated mycelium were indicative of an efficient anti-Saprolegnia activity of dioscin. The oxidative stress results showed that dioscin also accumulated reactive oxygen species excessively and increased total antioxidant and superoxide dismutase activity. These situations could render S. parasitica more vulnerable to oxidative damage. Additionally, when dioscin concentration was less than 2.0 mg L−1, the survival rate of embryos was more than 70%. Therefore, the use of dioscin could be a viable way of preventing and controlling saprolegniasis.
The use of dioscin could be a viable way of preventing and controlling saprolegniasis.
Oomycete infections in farmed fish are one of the most significant disease issues in salmonid aquaculture worldwide. In the present study, Saprolegnia spp. in different farmed fish species in Finland ...were identified, and the molecular epidemiology of especially Saprolegnia parasitica was examined. We analysed tissue samples from suspected oomycete‐infected salmonids of different life stages from a number of fish farms, as well as three wild salmonids. From collected oomycete isolates, the ITS1, 5.8S and ITS2 genomic regions were amplified, analysed phylogenetically and compared with corresponding sequences deposited in GenBank. Of the sequenced isolates, 91% were identified as S. parasitica. Isolates of yolk sac fry were identified as different Saprolegnia spp. Among the isolates from rainbow trout eggs Saprolegnia diclina dominated. In order to determine potential dominating clones among the S. parasitica, isolates were analysed using Multi Locus Sequence Typing (MLST). The results showed that one main clone contained the majority of the isolates. The MLST analysis showed four main sequence types (ST1–ST4) and 13 unique STs. This suggests that the Saprolegnia infections in farmed fish in Finland are not caused by different strains originating in the farm environment. Instead, one main clone of S. parasitica is present in Finnish fish farms.
Many amphibians are known to suffer embryonic die-offs as a consequence of an emergent disease known as 'Saprolegnia infections'. Thus far, the only species of Saprolegnia shown to be involved in ...natural infections is Saprolegnia ferax. In this study, we have isolated and characterized another Saprolegnia species responsible for 'Saprolegnia infections' on embryos of Bufo calamita in mountainous areas of central Spain. The strain was identified as belonging to Saprolegnia diclina based on morphological, physiological and molecular characters (sequencing of the internal transcribed region of ribosomal DNA). Zoospores of the new strain were able to infect embryos of Bufo calamita, and the symptoms observed were the same as those observed in natural infections. The results presented emphasize the need to carry out isolations and characterizations of the species and/or strains involved in this emergent disease. This will be important in order to design strategies to prevent the impact and spread of species (or strains) pathogenic to amphibians.
The ITS region of the rDNA gene was compared for Saprolegnia spp. in order to improve our understanding of nucleotide sequence variability within and between species of this genus, determine species ...composition in Canadian fin fish aquaculture facilities, and to assess the utility of ITS sequence variability in genetic marker development. From a collection of more than 400 field isolates, ITS region nucleotide sequences were studied and it was determined that there was sufficient consistent inter-specific variation to support the designation of species identity based on ITS sequence data. This non-subjective approach to species identification does not rely upon transient morphological features. Phylogenetic analyses comparing our ITS sequences and species designations with data from previous studies generally supported the clade scheme of Diéguez-Uribeondo et al. (2007) and found agreement with the molecular taxonomic cluster system of Sandoval-Sierra et al. (2014). Our Canadian ITS sequence collection will thus contribute to the public database and assist the clarification of Saprolegnia spp. taxonomy. The analysis of ITS region sequence variability facilitated genus- and species-level identification of unknown samples from aquaculture facilities and provided useful information on species composition. A unique ITS-RFLP for the identification of S. parasitica was also described.
•We determined Saprolegnia spp. composition for commercial aquaculture facilities.•Canadian ITS sequence data generally supports accepted species designations.•This study confirms the utility of ITS sequence data for species identification.•A diagnostic ITS-RFLP fingerprint was identified for Saprolegnia parasitica.
Saprolegniasis is an important disease in freshwater aquaculture, and is associated with oomycete pathogens in the genus Saprolegnia. Early detection of significant levels of Saprolegnia spp. ...pathogens would allow informed decisions for treatment which could significantly reduce losses. This study is the first to report the development of loop-mediated isothermal amplification (LAMP) for the detection of Saprolegnia spp. and compares it with quantitative PCR (qPCR). The developed protocols targeted the internal transcribed spacer (ITS) region of ribosomal DNA and the cytochrome C oxidase subunit 1 (CoxI) gene and was shown to be specific only to Saprolegnia genus. This LAMP method can detect as low as 10 fg of S. salmonis DNA while the qPCR method has a detection limit of 2 pg of S. salmonis DNA, indicating the superior sensitivity of LAMP compared to qPCR. When applied to detect the pathogen in water samples, both methods could detect the pathogen when only one zoospore of Saprolegnia was present. We propose LAMP as a quick (about 20-60 minutes) and sensitive molecular diagnostic tool for the detection of Saprolegnia spp. suitable for on-site applications.
Oomycetes of the genus Saprolegnia are responsible for severe economic losses in freshwater aquaculture. Following the ban of malachite green in food fish production, the demand for new treatments ...pushes towards the selection of more safe and environment‐friendly products. In the present work, in vitro activity of ten chemicals and three commercial products was tested on different strains of Saprolegnia, using malachite green as reference compound. The compounds were screened in agar and in water to assess the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC), respectively. Two strains of Saprolegnia parasitica and one isolate of Saprolegnia delica were tested in triplicate per each concentration. Among tested chemicals, benzoic acid showed the lowest MIC (100 ppm) followed by acetic acid, iodoacetic acid and copper sulphate (250 ppm). Sodium percarbonate was not effective at any tested concentration. Among commercial products, Virkon™S was effective in inhibiting the growth of the mycelium (MIC = MLC = 1,000 ppm). Actidrox® and Detarox® AP showed MIC = 5,000 and 1,000 ppm, respectively, while MLCs were 10‐fold lower than MICs, possibly due to a higher activity of these products in water. Similarly, a higher effectiveness in water was observed also for iodoacetic acid.
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