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This study aimed to investigate the potential of Chinese herbs in treating aquatic diseases. More particularly, the antibacterial properties and mechanisms of Chinese herbs and their monomers against Saprolegnia parasitica were investigated. In vitro antibacterial testing revealed that Cortex pseudolaricis exhibited significant antibacterial activity, with a minimum inhibitory concentration (MIC) of 0.98 mg/mL. The primary monomer responsible for this antibacterial effect was identified as pseudolaric acid B (PAB), with an MIC of 0.03 mg/mL. SEM and TEM analyses demonstrated that treatment with PAB resulted in structural damage to the cell wall and cell membrane of hyphae, leading to lysis of the cell wall and membrane of spores, organelle destruction, and vacuole formation within the cells. Analysis of the transcriptome and metabolome revealed that PAB disrupts amino acid, lipid, and nucleic acid metabolism in S. parasitica. This disruption impacts the biosynthesis and metabolism of various amino acids, including arginine, proline, glycine, serine, cysteine, methionine, glutamate, lysine, histidine, phenylalanine, tyrosine, and tryptophan. PAB also results in increased energy consumption and hindered energy generation in S. parasitica, as well as interference with the synthesis of membrane components such as DAG and phytosphingosine. Furthermore, PAB disrupts RNA, DNA, and ATP production in S. parasitica. Consequently, protein synthesis, energy supply, immune function and barrier structure in S. parasitica are weakened, and potentially leading to death. This study identifies potential antibacterial agents for environmentally friendly solutions for controlling fish saprolegniasis.

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Saprolegnia sp. is a pathogenic oomycete responsible for severe economic losses in aquaculture. To date, there is no treatment for its control that is effective and does not pose a threat to the environment and human health. In this research, two dihydrochalcones 1 and 2, and three chalcones 3-5, isolated from the resinous plant Adesmia balsamica, as well as their synthesized oxyalkylated derivatives 6-29 already reported and a new synthesized series of oxyalkylchalcones 30-35, were evaluated for their anti-saprolegnia activity and structure-activity relationship as potential control and treatment agents for strains of Saprolegnia parasitica and S. australis. Among the molecules tested, natural 2',4'-dihydroxychalcone (3) and new oxyalkylchalcone 34 were the most potent antisaprolegnia agents against both strains, even with better results than the commercial control bronopol. On the other hand, the structure-activity relationship study indicates that the contributions of steric and electrostatic fields are important to enhance the activity of the compounds, thus the presence of bulky substituents favors the activity.

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Saprolegnia parasitica is an oomycete responsible for a fish disease called saprolegniosis, which poses an economic and environmental burden on aquaculture production. In Saprolegnia, CHS5 of S. parasitica (SpCHS5) contains an N-terminal domain, a catalytic domain of the glycosyltransferase -2 family containing a GT-A fold, and a C-terminal transmembrane domain. No three-dimensional structure of SpCHS5 is reported yet disclosing the structural details of this protein. We have developed a structural model of full-length SpCHS5 and validated it by molecular dynamics simulation technique. From the 1 microsecond simulations, we retrieved the stable RoseTTAFold model SpCHS5 protein to explain characteristics and structural features. Furthermore, from the analysis of the movement of chitin in the protein cavity, we assumed that ARG 482, GLN 527, PHE 529, PHE 530, LEU 540, SER 541, TYR 544, ASN 634, THR 641, TYR 645, THR 641, ASN 772 residues as a main cavity lining site. In SMD analysis, we investigated the opening of the transmembrane cavity required for chitin translocation. The pulling of chitin from the internal cavity to the extracellular region was observed through steered molecular dynamics simulations. A comparison of the initial and final structures of chitin complex showed that there's a transmembrane cavity opening in the simulations. Overall, this present work will help us understand the structural and functional basis of CHS5 and design inhibitors against SpCHS5.Communicated by Ramaswamy H. Sarma.

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Host-associated microbial communities are an important determinant of individual fitness and have recently been highlighted as one of the factors influencing the success of invasive species. Invasive hosts introduce their microbes into the new environment, and then both the host and its associated microbes enter into a series of interactions with the native macroscopic and microscopic biota. As these processes are largely unexplored, we aimed to compare the exoskeletal microbial communities of co-occurring and phylogenetically related crayfish: the native narrow-clawed crayfish Pontastacus leptodactylus and the invasive signal crayfish Pacifastacus leniusculus from the recently invaded Korana River, Croatia. The results of high-throughput 16S rRNA sequencing showed that the exoskeletal microbiome of both species is very diverse, significantly influenced by the local environment and dominated by low abundance bacterial families from the phylum Proteobacteria. Furthermore, the exoskeletal microbiomes of the crayfish species differed significantly in the composition and abundance of Amplicon Sequence Variants (ASVs), suggesting that they are to some extent shaped by species-specific intrinsic factors, despite sharing a common habitat. However, over 95% of the bacterial genera associated with the exoskeleton were detected in the exoskeleton samples of both native and invasive crayfish. We paid particular attention to two known crayfish pathogens, Aphanomyces astaci and Saprolegnia parasitica, and find that both species carry low amounts of both pathogens. On the side, we find that a non-standard ddPCR protocol outperforms standard qPCR test for A. astaci under low concentration conditions. Taken together, our results indicate the possibility of bidirectional mixing and homogenisation of exoskeleton microbiome. As such, they can serve as a baseline in future detangling of the processes that act together to shape the microbiomes of co-occuring native and invasive congeners during biological invasions.

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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.

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Saprolegnia parasitica induces heavy mortality in aquaculture. The detection of S. parasitica is often time consuming and uncertain, making it difficult to manage the disease. We validated a previously published real-time quantitative PCR (qPCR) assay to confirm the presence of S. parasitica in fish and in water using environmental DNA (eDNA) quantification. Analytical sensitivity and specificity of the assay was assessed in silico, in vitro and the qPCR assay was compared with microbiological cultivation methods to detect and quantify S. parasitica in water samples from a controlled fish exposure experiment and from fish farms. Furthermore, we compared the use of an agar cultivation method and the qPCR assay to detect S. parasitica directly from mucus samples taken from the fish surface. The analytical sensitivity and specificity of the qPCR assay were high. The qPCR assay detected 100% of S. parasitica-positive water samples. In a field study, the qPCR assay and a microwell plate (MWP) enumeration method correlated significantly. Furthermore, the qPCR assay could be used to confirm the presence of S. parasitica in skin mucus. Thus, the qPCR assay could complement diagnostic methods in specifically detecting saprolegniosis in fish and used as a surveillance method for S. parasitica pathogen in aquaculture environments.

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Saprolegniasis, which is caused by Saprolegnia parasitica, leads to considerable economic losses. Recently, we showed that metalaxyl, bronopol and copper sulfate are good antimicrobial agents for aquaculture. In the current study, the efficacies of metalaxyl, bronopol and copper sulfate are evaluated by in vitro antimicrobial experiments, and the mechanism of action of these three antimicrobials on S. parasitica is explored using transcriptome technology. Finally, the potential target genes of antimicrobials on S. parasitica are identified by protein-protein interaction network analysis. Copper sulfate had the best inhibitory effect on S. parasitica, followed by bronopol. A total of 1771, 723 and 2118 DEGs upregulated and 1416, 319 and 2161 DEGs downregulated S. parasitica after three drug treatments (metalaxyl, bronopol and copper sulfate), separately. Additionally, KEGG pathway analysis also determined that there were 17, 19 and 13 significantly enriched metabolic pathways. PPI network analysis screened out three important proteins, and their corresponding genes were SPRG_08456, SPRG_03679 and SPRG_10775. Our results indicate that three antimicrobials inhibit S. parasitica growth by affecting multiple biological functions, including protein synthesis, oxidative stress, lipid metabolism and energy metabolism. Additionally, the screened key genes can be used as potential target genes of chemical antimicrobial drugs for S. parasitica.

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Saprolegnia parasitica is the most important pathogen under the genus, Saprolegnia which causes devastating oomycete diseases in freshwater fish. At present, the most common molecular method for identification of Saprolegnia species is sequencing of ribosomal DNA internal transcribed spacer (rDNA-ITS) region. In this study, a highly sensitive multiplex PCR targeting rDNA-ITS region and a hypothetical protein gene was developed using two sets of primer pair. In this PCR, two amplicons of different size of 750 bp and 365 bp are produced only in case of S. parasitica while other Saprolegnia species had single amplicon. This protocol could also differentiate Saprolegnia species from other fungus based on the size of rDNA-ITS region. The protocol does not require sequencing and can identify S. parasitica in a single reaction. Therefore, the multiplex PCR developed in this study may prove to be an easier, faster and cheaper molecular method for identification of S. parasitica.

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A controlled Saprolegnia parasitica infection model was used to challenge 1158 fish representing 105 pedigreed Atlantic salmon families to evaluate the possibility of selecting for Saprolegnia resistance in a commercial breeding programme. Fish were infected in five study tanks and observed for 40 days post-infection for lesion score and survival. Survival analysis of the top 10 resistant and bottom 10 susceptible families indicated that the hazard of dying following Saprolegnia infection was 1509% higher in susceptible families. In all fish, a 10 g increase in weight correlated with a 7.8% increase in the hazard of dying while sex did not affect mortality. Resistance to Saprolegnia was estimated to have a heritability of 0.25, indicating that selection is possible. Genetic and phenotypic correlations indicated that the 11-point scoring system, developed in this study to quantify Saprolegnia infection severity, had a high negative correlation with survival as days to mortality at ≥-0.922(±0.005), suggesting that the scoring method could help assess lesion development in studies where mortality is not the primary biological endpoint.

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Oomycete pathogens in freshwaters, such as Saprolegnia parasitica and Aphanomyces astaci, are responsible for fish/crayfish population declines in the wild and disease outbreaks in aquaculture. Although the formation of infectious zoospores in the laboratory can be triggered by washing their mycelium with natural water samples, the physico-chemical properties of the water that might promote sporulation are still unexplored. We washed the mycelia of A. astaci and S. parasitica with a range of natural water samples and observed differences in sporulation efficiency. The results of Partial Least Squares Regression (PLS-R) multivariate analysis showed that SAC (spectral absorption coefficient measured at 254 nm), DOC (dissolved organic carbon), ammonium-N and fluoride had the strongest positive effect on sporulation of S. parasitica, while sporulation of A. astaci was not significantly correlated with any of the analyzed parameters. In agreement with this, the addition of environmentally relevant concentrations of humic acid, an important contributor to SAC and DOC, to the water induced sporulation of S. parasitica but not of A. astaci. Overall, our results point to the differences in ecological requirements of these pathogens, but also present a starting point for optimizing laboratory protocols for the induction of sporulation.

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Saprolegnia infections are among the main parasitic diseases affecting farmed salmonids. The distribution and potential transfer of Saprolegnia spp. between farms and the natural environment has been scarcely investigated. Therefore, this work aimed to study the diversity and abundance of oomycete species in salmonid farms, tributary water, and effluent water systems. Four trout farms in Italy and two Atlantic salmon farms in Scotland were considered. In Italian farms, 532 isolates of oomycetes were obtained from fish and water, at upstream, inside, and downstream the farms. In Scottish farms, 201 oomycetes isolates were obtained from water outside the farm and from fish and water inside the farming units. Isolates were identified to the species level through amplification and sequencing of the ITS rDNA region. In Italy, S. parasitica was significantly more present in farmed than in wild fish, while in water it was more frequently isolated from the wild, particularly in effluent systems, not associated with more frequent isolation of S. parasitica in wild fish downstream the farm. In Scotland, S. parasitica was the most prevalent species isolated from fish, while isolates from water were mostly Pythium spp. with few S. parasitica isolates from upstream and downstream the farms.

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Saprolegnia parasitica, the causative agent of saprolegniosis in fish, and Aphanomyces astaci, the causative agent of crayfish plague, are oomycete pathogens that cause economic losses in aquaculture. Since toxic chemicals are currently used to control them, we aimed to investigate their inhibition by essential oils of sage, rosemary, and bay laurel as environmentally acceptable alternatives. Gas Chromatography-Mass Spectrometry (GC-MS) analysis showed that the essential oils tested were rich in bioactive volatiles, mainly monoterpenes. Mycelium and zoospores of A. astaci were more sensitive compared to those of S. parasitica, where only sage essential oil completely inhibited mycelial growth. EC50 values (i.e., concentrations of samples at which the growth was inhibited by 50%) for mycelial growth determined by the radial growth inhibition assay were 0.031-0.098 µL/mL for A. astaci and 0.040 µL/mL for S. parasitica. EC50 values determined by the zoospore germination inhibition assay were 0.007-0.049 µL/mL for A. astaci and 0.012-0.063 µL/mL for S. parasitica. The observed inhibition, most pronounced for sage essential oil, could be partly due to dominant constituents of the essential oils, such as camphor, but more likely resulted from a synergistic effect of multiple compounds. Our results may serve as a basis for in vivo experiments and the development of environmentally friendly methods to control oomycete pathogens in aquaculture.

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Copper-based fungicides have a long history of usage in agriculture and aquaculture. With the rapid development of metal-based nanoparticles, copper-based nanoparticles have attracted attention as a potential material for prevention and control of Saprolegnia parasitica. The present study investigated the effectiveness of copper/carbon core/shell nanoparticles (CCCSNs) and a commercial CCCSNs filter product (COPPERWARE®) against S. parasitica in a recirculating system. Results showed that the growth of agar plugs with mycelium was significantly suppressed after exposure to both CCCSNs powder and COPPERWARE® filters. Even the lowest concentration of CCCSNs used in our study (i.e., 100 mg/mL) exhibited significant inhibitory effects on S. parasitica. The smallest quantity of the filter product COPPERWARE® (3.75 × 3.7 × 1.2 cm, 2.58 g) used in our aquarium study also demonstrated significant inhibition compared with the control group. However, we observed leaching of copper into the water especially when larger quantities of COPPERWARE® were used. Water turbidity issues were also observed in tanks with the filter material. Besides these issues, which should be further investigated if the product is to be used on aquatic species sensitive to copper, CCCSNs has promising potential for water disinfection.

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Many Stramenopile species belonging to oomycetes from the genus Saprolegnia infect fish, amphibians, and crustaceans in aquaculture farms and natural ecosystems. Saprolegnia parasitica is one of the most severe fish pathogens, responsible for high losses in the aquaculture industry worldwide. Most of the molecules reported to date for the control of Saprolegnia infections either are inefficient or have negative impacts on the health of the fish hosts or the environment resulting in substantial economic losses. Until now, the whole proteome of S. parasitica has not been explored for a systematic screening of novel inhibitors against the pathogen. The present study was designed to develop a consensus computational framework for the identification of potential target proteins and their inhibitors and subsequent experimental validation of selected compounds. Comparative analysis between the proteomes of Saprolegnia, humans and fish species identified proteins that are specific and essential for the survival of the pathogen. The DrugBank database was exploited to select food and drug administration (FDA)-approved inhibitors whose high binding affinity to their respective protein targets was confirmed by computational modeling. At least six of the identified compounds significantly inhibited the growth of S. parasitica in vitro. Triclosan was found to be most effective with a minimum inhibitory concentration (MIC100) of 4 µg/ml. Optical microscopy showed that the inhibitors affect the morphology of hyphal cells, with hyper-branching being commonly observed. The inhibitory effects of the compounds identified in this study on Saprolegnia's mycelial growth indicate that they are potentially usable for disease control against this class of oomycete pathogens. Similar approaches can be easily adopted for the identification of potential inhibitors against other plant and animal pathogenic oomycete infections.

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Saprolegnia spp. water moulds are opportunistic pathogens that can cause economic losses to aquaculture. The diseases caused by them are difficult to control since use of the effective drug, malachite green oxalate, is no longer permitted in several regions (including the European Union and USA). To develop an effective control strategy, Saprolegnia isolates must be maintained in the laboratory. Cryopreservation is a useful solution for long-term maintenance; however, at present, there is no developed protocol for the cryopreservation of Saprolegnia spp. Here, we isolated and identified three Saprolegnia species, S. parasitica, S. australis and S. ferax, and developed a deep-freezing protocol that enables the long-term archiving of these species. The survival and growth rates of isolates kept at -80 °C for 3, 6, 9 and 12 months, were tested and compared among the species examined. Although the growth rates of frozen isolates were significantly lower than those of the control (i.e. non-frozen) isolates, the overall survival rate (>90%) indicated the effectiveness of the technique developed. Thus, the protocol developed appears to be a promising method for the long-term preservation of Saprolegnia isolates and may facilitate the creation of stock collections.

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Infectious diseases represent an important barrier to sustainable aquaculture development. Rearing density can substantially impact fish productivity, health and welfare in aquaculture, including growth rates, behaviour and, crucially, immune activity. Given the current emphasis on aquaculture diversification, stress-related indicators broadly applicable across species are needed. Utilising an interspecific comparative transcriptomic (RNAseq) approach, we compared gill gene expression responses of Atlantic salmon (Salmo salar) and Nile tilapia (Oreochromis niloticus) to rearing density and Saprolegnia parasitica infection. Salmon reared at high-density showed increased expression of stress-related markers (e.g. c-fos and hsp70), and downregulation of innate immune genes. Upon pathogen challenge, only salmon reared at low density exhibited increased expression of inflammatory interleukins and lymphocyte-related genes. Tilapia immunity, in contrast, was impaired at low-density. Using overlapping gene ontology enrichment and gene ortholog analyses, we found that density-related stress similarly impacted salmon and tilapia in key immune pathways, altering the expression of genes vital to inflammatory and Th17 responses to pathogen challenge. Given the challenges posed by ectoparasites and gill diseases in fish farms, this study underscores the importance of optimal rearing densities for immunocompetence, particularly for mucosal immunity. Our comparative transcriptomics analyses identified density stress impacted immune markers common across different fish taxa, providing key molecular targets with potential for monitoring and enhancing aquaculture resilience in a wide range of farmed species.

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A retrospective study was conducted using 250 clinical records of brown trout (Salmo trutta L.) with saprolegniosis by Saprolegnia parasitica, which had been collected from 8 rivers and 1 fish farm in the province of León (Spain). A geographic information system (GIS) was used to obtain skin lesion distribution patterns in males and females. Lesions in wild brown trout affected 15.31 ± 13.33% of the body surface, with a mean of 12.76 ± 6.56 lesions per fish. In addition, 51.23% of wild trout presented lesions with necrosis of the skin or fins. The pattern obtained when not distinguishing between sexes indicated that saprolegniosis lesions are mainly located above the lateral line and most frequently affect the dorsal cephalic region, the adipose fin, the peduncle and the caudal fin. However, differences were observed between males and females. Farmed trout presented a lower percentage of affected body surface (2.06 ± 4.36) and a lower number of lesions with and without necrosis because they received preventive treatment for saprolegniosis.

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Saprolegniasis is one of the most economical and ecologically harmful diseases in different species of fish. Low water temperature is one of the most important factors which increases stress and creates favourable conditions for the proliferation of Saprolegniasis. Therefore, the monitoring of water surface temperature (WST) is fundamental for a better understanding of Saprolegniasis. The objective of this study was to develop a predictive algorithm to estimate the probability of fish kills caused by Saprolegniasis in Río Tercero reservoir (Argentina). WST was estimated by Landsat 7 and 8 imagery using the Single-Channel method. Logistic regression was used to relate WST estimated from 2007 to 2017 with different episodes of fish kills by Saprolegniasis registered in the reservoir during this period of time. Results showed that the algorithm created with the first quartile (25th percentile) of the WST values estimated by Landsat sensors was the most suitable model to estimate Saprolegniasis in the studied reservoir.

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Saprolegnia parasitica is a pathogenic oomycete responsible for severe fish infections. Despite its low abundance in the cell wall of S. parasitica, chitin is essential for hyphal growth as the inhibition of its biosynthesis leads to highly reduced growth. Here we identified and characterized chitin synthases (CHS) from S. parasitica as potential targets for anti-oomycete drugs. Bioinformatics analyses allowed the identification of six different putative Chs genes in the genome of the pathogen. The total number of genes was confirmed by Southern blot analysis and their expression levels were determined by quantitative PCR. Four of the six Chs genes were expressed in the mycelium, while the two others exhibited undetectable levels of expression. The mycelium was highly sensitive to the addition of nikkomycin Z (NZ) in the culture medium, which led to a decreased amount of chitin in the cell wall by up to 40% in the conditions tested, and to the formation of abnormal branching structures in the hyphae. The presence of NZ increased the expression level of one of the genes, Chs3, suggesting that the corresponding product is compensating the disruption of chitin biosynthesis in the hyphae. In addition, the activity of isolated CHS was strongly inhibited by NZ in vitro. Altogether our data indicate the importance of CHS for the vegetative growth of S. parasitica and demonstrate that these enzymes represent promising targets for the control of diseases caused by oomycetes.