Final Report Summary - SAPRO (Sustainable Approaches to Reduce Oomycete (Saprolegnia) Infections in Aquaculture)
The overall objective of SAPRO was to create a training network for future researchers in an emerging area of central importance for European society and economy, the development of sustainable measures to control Saprolegniosis (http://youtu.be/t08vzE3KVVg (http://youtu.be/4J635ivHGYY)
Due to over-fishing of oceans, fish production has become increasingly dependent on fish farming for an adequate supply. Saprolegnia parasitica and Saprolegnia diclina are common fish pathogens and infection rates of fish and fish eggs with Saprolegniosis have increased in the fish farming industry worldwide since the ban of malachite green. Consequently, fish farmers need more sustainable measures to control Saprolegnia infections in aquaculture. Crucial for the development of new solutions to tackle Saprolegniosis is in-depth analysis of the biology of the pathogen and its hosts. For this purpose, the SAPRO training network used the expertise of its 8 partners to tackle the ecological, molecular, immunological and biochemical factors/traits underlying pathogen-host interactions and trained 10 Early Stage Researchers (ESRs) and 3 Early Researchers (ERs) in a whole range of complementary disciplines. The research was divided into eight interrelated scientific work packages (WPs), with each WP having a particular discipline focus.
Maintaining cell integrity is essential for viability and the enzymes responsible for this represent interesting potential targets to tackle the pathogen S. parasitica. The objectives of Biochemistry based WP1 was to identify such target enzymes for disease control and establish a mode of action of potential inhibitors. A comparative analysis of the cell wall composition of several pathogenic oomycetes, including Saprolegnia, Phytophthora and Aphanomyces species was completed. This unveiled the existence of 3 clearly different cell wall types. A quantitative proteomics approach identified a total of 1655 plasma membrane proteins from hyphal cells. Several 1,3-β-glucan synthases and transglycosylases were found to be highly enriched in plasma membrane microdomains, pointing to a functional specialization. Several of these proteins were therefore targeted for disease control studies. For this, compounds were identified that affected growth of S. parasitica, screening then selected the compounds that specifically affected the targeted proteins. In this way five compounds were identified that were directed against different cell wall synthesizing enzymes in S. parasitica, which is a very promising lead for further development. As part of the genomics approach in WP2, the genomes of S. parasitica strain CBS 223.65 and N12 and S. diclina strain VS20 were fully sequenced, annotated and made available to the public. This enabled us to develop much needed molecular tools for S. parasitica to identify and study the function of potential virulence genes. The involvement of a fibronectin protein in cyst attachment to the host and the involvement of the melanin in protecting S. parasitica from host defence mechanisms, were studied by gene knockdown via interference RNA providing potential targets for disease control.
A further target for disease control was identified through the search for morphological markers within WP3 which looked at the Ecology and Biodiversity of S. parasitica. Spines of cysts were identified and described and the molecular techniques developed in WP2 facilitated functional characterization of this crucial morphological marker. New markers were developed using RAPD-PCR, AFLP-PCR, Microsatellites, and PCR based species-specific ITS markers and were applied to identify species and populations. A new pathogenic species to salmonid eggs, Saprolegnia australis, was found and its resistance to bronopol treatment characterized. The use of all these molecular markers has been looked at in two case-studies in Spain and Baltic countries to help decision making in management strategies for local fisheries. In addition a unique culture collection of Saprolegniales was built with 1000 specimens and a Saprolegnia DNA bank created containing more than 3000 extractions of Saprolegniales which represented most of the Biodiversity of this group. This has allowed us to make the first molecular taxonomy of Saprolegnia using MOTUs (molecular operational taxonomic units) which greatly facilitates a more rapid and precise identification of economically important species of Saprolegnia.
The pathology and infectivity of Saprolegnia species in fish and fish eggs was investigated in WP4. Saprolegnia parasitica and Saprolegnia diclina hyphae were shown to penetrate intact chorion of Atlantic salmon (Salmo salar L.) non-eyed eggs. We were also able to correlate a thicker chorion with protection against Saprolegnia infection in Atlantic salmon. These studies provide essential information regarding possible modes of resistance to Saprolegniosis and would possibly allow breeding for resistance.
A bioinformatical approach to virulence and molecular pathogen-host interactions was employed in WP5, to search for ‘effector proteins’ that are required to establish a successful infection. The genome of S. parasitica was searched and from 30 potential candidates, protein SpHtp3 was identified and characterised. Protein SpHtp3 was found to have both self-translocation and nuclease activities in one molecule, the first time an oomycete protein has been reported to possess this. The translocation process of SpHtp3 was found to be pathogen-independent, but its release from vesicles was pathogen-dependent. Once inside the host cells, we hypothesised SpHtp3 may interfere with the RNA metabolism to promote infection or to provide nutrients. In a broader context, the work on SpHtp3 has helped us to understand the oomycete (especially S. parasitica) infection process and how proteins are targeted for host cell uptake. The bioinformatic data generated will assist in the identification and characterisation of more SpHtp3 - like proteins that are able to enter host cells.
The ability of Saprolegnia to establish a successful infection is also determined by the immune status of the host. WP6 examined the host defence and a detailed immune profile of Atlantic salmon during Saprolegnia parasitica infection was completed. We demonstrated that the Atlantic salmon immune system responds with a strong inflammatory response together with the activation of the innate immunity (antimicrobial peptides) and acute phase proteins (Fig 2, A,B). Specifically it was found that prostaglandin-E2 (PGE2) triggered a moderate inflammatory response while suppressing cytokines associated with adaptive immunity and Igs. (fig2 C). Two host-targeting effector proteins identified by ESR6, SpHtp-1 and SpHtp-3 were shown to interact with rainbow trout head kidney leucocytes and using red-fluorescence tagged proteins (mRFP) it was shown that both SpHtp1 and SpHtp3 translocated in a macrophage-specific manner (Fig 2D). We also discovered that S. parasitica was capable of secreting proteases that have the ability to degrade rainbow trout immunoglobulin M (IgM) (Fig 2E).
Figure 2 Response of Host immune system
Disease management was addressed in WP7. One approach was to investigate biocontrol of Saprolegnia. To this end, beneficial microorganisms from salmon egg cultures were sought that could suppress the growth and/or infectious activity of Saprolegnia. Meta-taxonomic analyses revealed that Atlantic salmon eggs from hatcheries are home to diverse oomycete, fungal, bacterial and archaeal communities. While virulent Saprolegnia isolates were found in all salmon egg samples, a low incidence of Saprolegniosis was strongly correlated with a high richness and abundance of specific commensal Actinobacteria, with the genus Frondihabitans (Microbacteriaceae) effectively inhibiting attachment of Saprolegnia to salmon eggs
Figure 3 Diseased (left) and healthy (right) salmon egg samples
These fundamental insights into microbial landscapes of fish eggs may provide new sustainable means to mitigate Saprolegniosis and possibly other diseases. The final scientific work package in the SAPRO program, WP8 brought the fundamental findings together for drug and vaccine development and testing. The ability of Saprolegnia isolates to form biofilms and subsequently produce infective motile zoospores was confirmed in vitro. This was a consideration when testing the effectiveness of different chemicals, as this enables Saprolegnia to resist different chemicals used for its control. Our industrial partner was involved in the assessment and selection of several candidate substances identified in WP1-7: a vaccine candidate; biocontrol candidates and chemical candidates. Vaccination experiments gave insights into the activity of the candidate protein and the interaction between the fish immune system and the pathogen. Challenge systems were optimized for testing prophylaxis and treatment against Saprolegnia of salmon and trout. A test system for testing prophylaxis and treatment against Saprolegnia for salmonid ova was also developed. Potential impact and societal implications of project Research results have been disseminated through scientific publications in international scientific journals and industry specific journals as well as through multiple methods of communications within the SAPRO program, industry and stake holders, as well as at top-level international conferences. It is expected that the multidisciplinary background that the fellows have acquired will facilitate their recruitment in academic research groups and also in industry. State-of-the-art technologies and methodologies were used to identify potential disease targets and develop novel control measures. Our industrial partner and several consortium members will investigate further some of the best leads in order to develop into products that may be used to control Saprolegniosis in the aquaculture industry in the future.
Due to over-fishing of oceans, fish production has become increasingly dependent on fish farming for an adequate supply. Saprolegnia parasitica and Saprolegnia diclina are common fish pathogens and infection rates of fish and fish eggs with Saprolegniosis have increased in the fish farming industry worldwide since the ban of malachite green. Consequently, fish farmers need more sustainable measures to control Saprolegnia infections in aquaculture. Crucial for the development of new solutions to tackle Saprolegniosis is in-depth analysis of the biology of the pathogen and its hosts. For this purpose, the SAPRO training network used the expertise of its 8 partners to tackle the ecological, molecular, immunological and biochemical factors/traits underlying pathogen-host interactions and trained 10 Early Stage Researchers (ESRs) and 3 Early Researchers (ERs) in a whole range of complementary disciplines. The research was divided into eight interrelated scientific work packages (WPs), with each WP having a particular discipline focus.
Maintaining cell integrity is essential for viability and the enzymes responsible for this represent interesting potential targets to tackle the pathogen S. parasitica. The objectives of Biochemistry based WP1 was to identify such target enzymes for disease control and establish a mode of action of potential inhibitors. A comparative analysis of the cell wall composition of several pathogenic oomycetes, including Saprolegnia, Phytophthora and Aphanomyces species was completed. This unveiled the existence of 3 clearly different cell wall types. A quantitative proteomics approach identified a total of 1655 plasma membrane proteins from hyphal cells. Several 1,3-β-glucan synthases and transglycosylases were found to be highly enriched in plasma membrane microdomains, pointing to a functional specialization. Several of these proteins were therefore targeted for disease control studies. For this, compounds were identified that affected growth of S. parasitica, screening then selected the compounds that specifically affected the targeted proteins. In this way five compounds were identified that were directed against different cell wall synthesizing enzymes in S. parasitica, which is a very promising lead for further development. As part of the genomics approach in WP2, the genomes of S. parasitica strain CBS 223.65 and N12 and S. diclina strain VS20 were fully sequenced, annotated and made available to the public. This enabled us to develop much needed molecular tools for S. parasitica to identify and study the function of potential virulence genes. The involvement of a fibronectin protein in cyst attachment to the host and the involvement of the melanin in protecting S. parasitica from host defence mechanisms, were studied by gene knockdown via interference RNA providing potential targets for disease control.
A further target for disease control was identified through the search for morphological markers within WP3 which looked at the Ecology and Biodiversity of S. parasitica. Spines of cysts were identified and described and the molecular techniques developed in WP2 facilitated functional characterization of this crucial morphological marker. New markers were developed using RAPD-PCR, AFLP-PCR, Microsatellites, and PCR based species-specific ITS markers and were applied to identify species and populations. A new pathogenic species to salmonid eggs, Saprolegnia australis, was found and its resistance to bronopol treatment characterized. The use of all these molecular markers has been looked at in two case-studies in Spain and Baltic countries to help decision making in management strategies for local fisheries. In addition a unique culture collection of Saprolegniales was built with 1000 specimens and a Saprolegnia DNA bank created containing more than 3000 extractions of Saprolegniales which represented most of the Biodiversity of this group. This has allowed us to make the first molecular taxonomy of Saprolegnia using MOTUs (molecular operational taxonomic units) which greatly facilitates a more rapid and precise identification of economically important species of Saprolegnia.
The pathology and infectivity of Saprolegnia species in fish and fish eggs was investigated in WP4. Saprolegnia parasitica and Saprolegnia diclina hyphae were shown to penetrate intact chorion of Atlantic salmon (Salmo salar L.) non-eyed eggs. We were also able to correlate a thicker chorion with protection against Saprolegnia infection in Atlantic salmon. These studies provide essential information regarding possible modes of resistance to Saprolegniosis and would possibly allow breeding for resistance.
A bioinformatical approach to virulence and molecular pathogen-host interactions was employed in WP5, to search for ‘effector proteins’ that are required to establish a successful infection. The genome of S. parasitica was searched and from 30 potential candidates, protein SpHtp3 was identified and characterised. Protein SpHtp3 was found to have both self-translocation and nuclease activities in one molecule, the first time an oomycete protein has been reported to possess this. The translocation process of SpHtp3 was found to be pathogen-independent, but its release from vesicles was pathogen-dependent. Once inside the host cells, we hypothesised SpHtp3 may interfere with the RNA metabolism to promote infection or to provide nutrients. In a broader context, the work on SpHtp3 has helped us to understand the oomycete (especially S. parasitica) infection process and how proteins are targeted for host cell uptake. The bioinformatic data generated will assist in the identification and characterisation of more SpHtp3 - like proteins that are able to enter host cells.
The ability of Saprolegnia to establish a successful infection is also determined by the immune status of the host. WP6 examined the host defence and a detailed immune profile of Atlantic salmon during Saprolegnia parasitica infection was completed. We demonstrated that the Atlantic salmon immune system responds with a strong inflammatory response together with the activation of the innate immunity (antimicrobial peptides) and acute phase proteins (Fig 2, A,B). Specifically it was found that prostaglandin-E2 (PGE2) triggered a moderate inflammatory response while suppressing cytokines associated with adaptive immunity and Igs. (fig2 C). Two host-targeting effector proteins identified by ESR6, SpHtp-1 and SpHtp-3 were shown to interact with rainbow trout head kidney leucocytes and using red-fluorescence tagged proteins (mRFP) it was shown that both SpHtp1 and SpHtp3 translocated in a macrophage-specific manner (Fig 2D). We also discovered that S. parasitica was capable of secreting proteases that have the ability to degrade rainbow trout immunoglobulin M (IgM) (Fig 2E).
Figure 2 Response of Host immune system
Disease management was addressed in WP7. One approach was to investigate biocontrol of Saprolegnia. To this end, beneficial microorganisms from salmon egg cultures were sought that could suppress the growth and/or infectious activity of Saprolegnia. Meta-taxonomic analyses revealed that Atlantic salmon eggs from hatcheries are home to diverse oomycete, fungal, bacterial and archaeal communities. While virulent Saprolegnia isolates were found in all salmon egg samples, a low incidence of Saprolegniosis was strongly correlated with a high richness and abundance of specific commensal Actinobacteria, with the genus Frondihabitans (Microbacteriaceae) effectively inhibiting attachment of Saprolegnia to salmon eggs
Figure 3 Diseased (left) and healthy (right) salmon egg samples
These fundamental insights into microbial landscapes of fish eggs may provide new sustainable means to mitigate Saprolegniosis and possibly other diseases. The final scientific work package in the SAPRO program, WP8 brought the fundamental findings together for drug and vaccine development and testing. The ability of Saprolegnia isolates to form biofilms and subsequently produce infective motile zoospores was confirmed in vitro. This was a consideration when testing the effectiveness of different chemicals, as this enables Saprolegnia to resist different chemicals used for its control. Our industrial partner was involved in the assessment and selection of several candidate substances identified in WP1-7: a vaccine candidate; biocontrol candidates and chemical candidates. Vaccination experiments gave insights into the activity of the candidate protein and the interaction between the fish immune system and the pathogen. Challenge systems were optimized for testing prophylaxis and treatment against Saprolegnia of salmon and trout. A test system for testing prophylaxis and treatment against Saprolegnia for salmonid ova was also developed. Potential impact and societal implications of project Research results have been disseminated through scientific publications in international scientific journals and industry specific journals as well as through multiple methods of communications within the SAPRO program, industry and stake holders, as well as at top-level international conferences. It is expected that the multidisciplinary background that the fellows have acquired will facilitate their recruitment in academic research groups and also in industry. State-of-the-art technologies and methodologies were used to identify potential disease targets and develop novel control measures. Our industrial partner and several consortium members will investigate further some of the best leads in order to develop into products that may be used to control Saprolegniosis in the aquaculture industry in the future.