Development and comparison of loop-mediated isothermal amplification with quantitative PCR for the specific detection of Saprolegnia spp.

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.

First Record of the Water Mold Achlya bisexualis (Saprolegniaceae) Isolated from Ornamental Fish in South Africa.

The order Saprolegniales (Class Oomycota) is a group of fungus-like eukaryotic microorganisms that have been associated with infections in fish and fish eggs. Infections with microorganisms from this order are clearly evident because they have a cotton wool-like appearance. The aim of this study was to characterize and identify an oomycete that was isolated from the eye of an Orange Blotched Peacock Cichlid Aulonacara sp. A sample of cotton wool-like mycelia was isolated and single-spore isolations were conducted. Molecular characterization and phylogenetic analysis of the ITS1-5.8-ITS2 rDNA region for all isolates were used for species identification. Following molecular identification, one isolate was used to culture and characterize the reproductive structures. Physiological characterization entailed incubating the isolate on potato dextrose agar (PDA) at five different temperatures, ranging from 5°C to 25°C, to monitor growth rates. A multiple sequence alignment showed 100% similarity between all of the single-spore isolates and alignment with other Achlya bisexualis strains. Long, coarse hyphae with zoosporangia and gemmae typical of the order Saprolegniales were observed with an optimal growth rate at 25°C. The oomycete that was isolated from an Orange Blotched Peacock Cichlid was identified as A. bisexualis, the first record of this species in South Africa.

Saprolegnia molecular phylogeny among farmed teleosts in Nova Scotia, Canada.

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.

Multilocus sequence typing reveals clonality in Saprolegnia parasitica outbreaks.

The molecular epidemiology of fish pathogen Saprolegnia parasitica is still largely unknown. We developed a multilocus sequence typing scheme based on seven housekeeping genes to characterize 77 S. parasitica strains isolated from different fish host species at different times and from different geographic areas in Switzerland between 2015 and 2017. Ten different diploid sequence types (DSTs) were identified. The majority (52%) of outbreaks in Switzerland seemed to be caused by one genotype, namely DST3, which was recovered from farm-raised and wild-caught fish in all the geographic areas and river basins included in the study. DST3 was also recovered from the rivers Bienne (eastern France) and Doubs, where the episodes of massive mortality due to saprolegniosis started in 2009. Another genotype (DST7) showed, to a lesser extent, a distribution across different river basins, while eight DSTs were unique to a defined geographic area or river basin. The occurrence of sporadic DSTs indicates a certain degree of diversity within S. parasitica in the environment. The wide distribution of DST3 suggests that a clonal population may have spread in eastern France and Switzerland across geographic barriers, with strong implications for the management of both captive and wild fish populations.

Saprolegniosis in Nile Tilapia: Identification, Molecular Characterization, and Phylogenetic Analysis of Two Novel Pathogenic Saprolegnia Strains.

Saprolegniosis is a fungal infection that leads to huge economic losses in tilapia aquaculture. Saprolegnia spp. are usually implicated as the etiological agents, but their identification is sometimes troublesome and confusing. In this study, two Saprolegnia strains (ManS22 and ManS33) were isolated from Nile Tilapia Oreochromis niloticus suffering from saprolegniosis. Both isolates were characterized morphologically and from internal transcribed spacer (ITS) sequence data. Additionally, both strains were tested for pathogenicity, and they were highly pathogenic and caused cumulative mortalities of 88.9% and 95.6%, respectively. Initially, the two strains were identified, by morphology of sexual and asexual stages, as members of the genus Saprolegnia. For more definitive identification and characterization, the ITS region of the ribosomal RNA genes was amplified and sequenced, and sequences were compared with other known sequences in GenBank. A phylogenetic tree constructed using the neighbor-joining method revealed that the two strains fell into two clusters within the species Saprolegnia parasitica. Cluster 1 included the ManS33 strain and cluster 2 the ManS22 strain. Cluster 1 grouped the ManS33 strain with other S. parasitica stains and shared 97-99% sequence similarity. Cluster 2 contained only the ManS22 strain and shared 93-94% similarity to several reference sequences of S. parasitica strains. Therefore, our findings suggest that ManS22 represents a newly described strain of S. parasitica. Received April 19, 2016; accepted October 27, 2016.

A Comprehensive Protocol for Improving the Description of Saprolegniales (Oomycota): Two Practical Examples (Saprolegnia aenigmatica sp. nov. and Saprolegnia racemosa sp. nov.).

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.

Saprolegnia species in Norwegian salmon hatcheries: field survey identifies S. diclina sub-clade IIIB as the dominating taxon.

Saprolegnia isolates within the recognized clades encompassing the taxa S. parasitica and S. diclina act as opportunist and aggressive pathogens to both fish and their eggs. They are responsible for significant economic losses in aquaculture, particularly in salmonid hatcheries. However, the identity, distribution and pathogenic significance of involved species often remain unexplored. In this study, 89 Saprolegnia isolates were recovered from water, eggs and salmon tissue samples that originated from salmon (Salmo salar) hatcheries along the coast of Norway. The cultures were characterized morphologically and molecularly in order to provide an overview of the species composition of Saprolegnia spp. present in Norwegian salmon hatcheries. We demonstrate that S. diclina clearly dominated and contributed to 79% of the recovered isolates. Parsimony analyses of the nuclear ribosomal internal transcribed spacer (ITS) region split these isolates into 2 strongly supported sub-clades, S. diclina sub-clade IIIA and IIIB, where sub-clade IIIB accounted for 66% of all isolates. A minor portion of the isolates constituted other taxa that were either conspecific or showed strong affinity to S. parasitica, S. ferax, S. hypogyna and Scoliolegnia asterophora. The unique sub-clade IIIB of S. diclina was most prevalent in water and salmon eggs, while S. parasitica isolates were more frequently isolated from post hatching stages. The study demonstrated that morphological criteria in many cases were insufficient for species delimitation due to lack of sexual structures or incoherent morphological expression of such features within the tested replicates.

Species composition of the genus Saprolegnia in fin fish aquaculture environments, as determined by nucleotide sequence analysis of the nuclear rDNA ITS regions.

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.

Species identification in the genus Saprolegnia (Oomycetes): defining DNA-based molecular operational taxonomic units.

The lack of a robust taxonomy in the genus Saprolegnia is leading to the presence of incorrectly named isolates in culture collections and of an increasing number of misassigned sequences in DNA databases. Accurate species delimitation is critical for most biological disciplines. A recently proposed approach to solve species delimitation (taxonomic diagnosis system) of difficult organisms is the definition of molecular operational taxonomic units (MOTUs). We have used 961 sequences of nrDNA ITS from culture collections (461 sequences) and GenBank (500 sequences), to perform phylogenetic and clustering optimization analyses. As result, we have identified 29 DNA-based MOTUs in agreement with phylogenetic studies. The resulting molecular clusters support the validity of 18 species of Saprolegnia and identify 11 potential new ones. We have also listed a number of incorrectly named isolates in culture collections, misassigned species names to GenBank sequences, and reference sequences for the species. We conclude that GenBank represents the main source of errors for identifying Saprolegnia species since it possesses sequences with misassigned names and also sequencing errors. The presented taxonomic diagnosis system might help setting the basis for a suitable identification of species in this economically important genus.

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.

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.

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.

Identification of an isolate of Saprolegnia ferax as the causal agent of saprolegniosis of yellow catfish (Pelteobagrus fulvidraco) eggs.

Saprolegnia species have been implicated for significant fungal infections of both living and dead fish as well as their eggs. In the present study, an oomycete water mould (strain HP) isolated from yellow catfish (Peleobagrus fulvidraco) eggs suffering from saprolegniosis was characterized both morphologically and from ITS sequence data. It was initially identified as a Saprolegnia sp. isolate based on its morphological features. The constructed phylogenetic tree using neighbour joining method indicated that the HP strain was closely related to Saprolegnia ferax strain Arg4S (GenBank accession no. GQ119935), that had previously been isolated from farming water samples in Argentina. In addition, the zoospore numbers of strain HP were markedly influenced by a variety of environmental variables including temperature, pH, formalin and dithiocyano-methane. Its zoospore formation was optimal at 20 °C and pH 7, could be well inhibited by formalin and dithiocyano-methane above 5 mg/L and 0.25 mg/L, respectively. To our knowledge, this is the first report on the S. ferax infection in the hatching yellow catfish eggs.

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.

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.

A novel Salifa-Saprolegnia association.

During the period from December 2006 to March 2007, about 1000 freshwater leeches, Salifa delicata, were collected from Al-sont canal, adjacent to Assiut city, Egypt. In the laboratory, 96% of Salifa delicata showed signs of oomycotal infection (cotton-wool like appearance radiating out in whorled pattern) and died within 3 days. Direct microscopy and culture proved Saprolegnia hypogyna to be the pathogen. Histopathological studies showed necrotic lesions, destruction of cuticle, epidermis, dermis, muscle layers, botryoidal tissue and even the gut with the oomycete hyphae penetrating the damaged tissues. To the best of the authors' knowledge, this represents the first record of a novel association between the leech, Salifa delicata, and the oomycete, Saprolegnia hypogyna, but the second report on the histopathology of saprolegniasis within leeches.

Saprolegnia brachydanis, a new oomycete isolated from zebra fish.

Saprolegnia brachydanis is described from zebra fish (Brachydanio rerio) in Wuhan, Hubei Province, China. The species is illustrated and compared with other species of the genus. The distinctive characteristics of S. brachydanis are the production of glomerulate oogonia wrapped around by predominantly monoclinous antheridia which can be up to eight in one oogonium. The oogonial stalks are short, straight, or curved and the antheridia, twisted, can enwind one or more oogonia. The oospores cannot mature or easily abort. Morphological features of the oomycete and the ITS sequence of its rDNA as well as the comparison with related species are discussed in this article.

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.

Saprolegnia diclina: another species responsible for the emergent disease 'Saprolegnia infections' in amphibians.

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.

Species boundaries within Saprolegnia (Saprolegniales, Oomycota) based on morphological and DNA sequence data.

Saprolegnia is a common and widespread genus of Oomycetes, however species identifications are difficult and uncertain. To test whether keys based on morphological characters could identify species as determined by molecular characters we determined partial DNA sequences for the 28S rRNA gene and the complete internal transcribed spacer (ITS) region for 55 isolates belonging to Saprolegnia and one isolate of Protoachlya hypogyna that exhibited saprolegnoid zoospore discharge in water culture. Phylogenetic analyses of the combined sequence data yielded 10 robustly supported clades that probably represent separate species. Morphological analyses of all isolates revealed that each DNA-based clade could be delimited from others by autapomorphic or unique combinations of morphological character states but not without employing several features previously not used at the species level. Taxonomic implications of these results are discussed and recommendations for less equivocal characterization of new Saprolegnia species are made.