Development of a 3D spheroid cell culture system from fish cell lines for in vitro infection studies: Evaluation with Saprolegnia parasitica.

Understanding the ways in which pathogens infect host cells is essential to improve and develop new treatment strategies. This study aimed to generate a novel in vitro infection model by establishing a reproducible 3D spheroid cell culture system that may lead to a reduced need for animals in fish disease research. 2D models (commonly cell lines) cannot replicate many key conditions of in vivo infections, but 3D spheroids have the potential to provide bridging technology between in vivo and in vitro systems. 3D spheroids were generated using cells from rainbow trout (Oncorhynchus mykiss) cell lines, RTG-2 and RTS-11. The RTG-2 spheroids were tested for their potential to be infected upon exposure to Saprolegnia parasitica spores. Positive infiltration of mycelia into the spheroids was verified by confocal microscopy. As a closer analogue of in vivo conditions encountered during infection, the straightforward model developed in this study shows promise as an additional tool that can be used to further our understanding of host-pathogen interactions for Saprolegnia and possibly a variety of other fish pathogens.

Cryopreservation of three Saprolegnia species (Oomycota): Preliminary evidence for the long-term archiving of water mould species.

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.

Immunity and growth improvement of rainbow trout (Oncorhynchus mykiss) fed dietary nettle (Urtica dioica) against experimental challenge with Saprolegnia parasitica.

In this study, effects of nettle (Urtica dioica) on growth, immunity, and gene expressions were examined in rainbow trout after an 8-week feeding period. A total of 264 juvenile rainbow trout (10.72 ± 0.55 g) were selected and stocked randomly in 12 aquaria. Nettle powder was added to the fish feed at three doses, 0.5,1 and 1.5% served as treatments. At the end of 8-week feeding period, the fish were exposed to Saprolegnia parasitica for 3 weeks. Results showed that all treatments fed with nettle diets exhibited significant increases in weight gain and SGR, and decreased FCR compared to the control. Feeding the fish with dietary nettle resulted in significant rises in blood indices and non-specific immunity in comparison with the control. Furthermore, fish fed 0.5% of dietary nettle showed significantly increased expressions of TNF-α, IL-1b, IL-6 and IL-8 genes following 8 weeks of feeding. A significant reduction in mortality rate was observed in the fish treated with 0.5% of nettle compared to the control following challenging with S. parasitica. Our observations indicate that the use of 0.5% nettle powder in rainbow trout diet can improve growth and immunity parameters as well as fish resistance against S. parasitica contamination.

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.

Distribution patterns of saprolegniosis cutaneous lesions in wild and farmed brown trout (Salmo trutta L.) obtained using a geographic information system (GIS).

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.

Colorimetric assay for the in vitro evaluation of Saprolegnia biofilm inhibitors.

A quantitative and reproducible 96-well microtiter method that is easily adaptable for the screening of Saprolegnia biofilm inhibitors is described. As opposed to other methods previously developed for the screening of Saprolegnia inhibitors on spore germination or mycelial growth, this technique is of particular significance as it investigates potential inhibitors against surface-attached mycelial mats of Saprolegnia spp. (biofilm). In this study, we have investigated the effects of propionic acid (PPA) on reducing the viability of induced Saprolegnia biofilms using colorimetric MTS assay based on the reduction of tetrazolium salts. Viability of Saprolegnia hyphae in treated biofilms was reduced significantly following treatment with different PPA concentrations. The effect was enhanced after combining each of the tested PPA concentrations with 500 mg/L of boric acid (BA). However, the percentage of non-viable hyphae was still higher in 200 mg L-1 bronopol-treated biofilms (positive control) following 6- and 12-hr exposure. Similar results were observed using other recently described fluorescence-based assays for viability.

Remote sensing application to estimate fish kills by Saprolegniasis in a reservoir.

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.

T cell receptor (TCR) α and ß genes of loach (Misgurnus anguillicaudatus): Molecular cloning and expression analysis in response to bacterial, parasitic and fungal challenges.

In vertebrates, the T cell receptor (TCR) plays a crucial role in immune system. To date, the roles of fish TCRs in response to pathogen infection are still poorly understood. In the present study, we firstly cloned and identified the TCRα and TCRß from dojo loach (Misgurnus anguillicaudatus) by RACE approaches. The full-length cDNAs of Ma-TCRα and Ma-TCRß include an open reading frame (ORF) of 723 and 879 bp encoding a polypeptide of 241 and 293 amino acids, respectively. Structural analysis indicated that Ma-TCRα and Ma-TCRß had a signal peptide, IgV domain, IgC domain, a connecting peptide (CPS), a transmembrane region (TM) and a cytoplasmic (CYT), which are similar to their counterparts described in other teleost. Phylogenetic analysis supported that Ma-TCR Cα and Ma-TCR Cß were closely related to the Cα and Cß region of Cyprinidae family, respectively. Transcriptional expression analysis indicated that Ma-TCRα and Ma-TCRß mRNAs were ubiquitously expressed in a wide array of tissues and most abundantly found in skin, brain, kidney, gill and spleen. The expression patterns of Ma-TCRα and Ma-TCRß after bacteria (F. columnare G4), parasite (Ichthyophthirius multifiliis) and fungus (Saprolegnia) infection were detected by qRT-PCR. Additionally, the morphological changes of gill and skin following the three infection models were investigated. The results clearly indicated that Ma-TCRα and Ma-TCRß was significant up-regulated not only in spleen and kidney, but also in skin and gill. In summary, our present findings suggested that Ma-TCRα and Ma-TCRß might play significantly roles in the modulation of immune response and protect loach from different pathogens infection.

Stress-immune responses and DNA protection function of thioredoxin domain containing 12 in zebrafish (Danio rerio).

Proteins with dithiol-disulfide oxidoreductase catalytic domain are well known for their capacity in the cellular redox homeostasis. In this study, we characterized the zebrafish thioredoxin domain containing 12 (Zftxndc12) gene, analyzed the transcriptional responses and studied the functional properties of its recombinant protein. Full-length cDNA of Zftxndc12 consists 519 bp coding region encoding 172 amino acids (AA) including the signal peptide. Highly consensus active motif (65WCGAC69) and probable ER retrieval motif (169GDEL172) were identified. Ubiquitous expression of Zftxndc12 mRNA was observed from one cell to juvenile stage as well as different organs of adult zebrafish. Moreover, whole mount in situ hybridization (WISH) results showed a higher expression of Zftxndc12 in primordial gills, central nerves system and eye. The tissue specific expression analysis (by qRT-PCR) also showed the highest expression in gills followed by brain in adult zebrafish. In larvae, up-regulated Zftxndc12 mRNA expression upon exposure to H2O2,Edwardsiella tarda and Saprolegnia parasitica suggests that it may involve in both stress and immune responses. Moreover, transcriptional expression of Zftxndc12 was up-regulated upon Streptococcus iniae challenge in gills of adult zebrafish. The recombinant ZfTxndc12 (rZfTxndc12) was overexpressed, purified and tested for its biological activities. Results revealed that rZfTxndc12 is able to reduce the DNA damage and detoxify the H2O2 toxicity in concentration dependent manner. Overall results suggest that Zftxndc12 is important antioxidant and immune functional member of the host defense system in zebrafish.

Transcriptomic response to parasite infection in Nile tilapia (Oreochromis niloticus) depends on rearing density.

BACKGROUND: Captive animal populations, be it for food production or conservation programmes, are often maintained at densities far beyond those in natural environments, which can have profound effects on behaviour, immune and stress levels, and ultimately welfare. How such alterations impact transcriptional responses to pathogen infection is a 'different kettle of fish' and remains poorly understood. Here, we assessed survival and gene expression profiles of infected fish reared at two different densities to elucidate potential functional genomic mechanisms for density-related differences in disease susceptibility. RESULTS: Utilising a whole-transcriptome sequencing (RNAseq) approach, we demonstrate that rearing density in tilapia (Oreochromis niloticus) significantly impacts susceptibility to the oomycete Saprolegnia parasitica, via altered transcriptional infection responses. Tilapia held at low densities have increased expression of genes related to stress, likely due to increased aggressive interactions. When challenged with Saprolegnia, low-density fish exhibit altered expression of inflammatory gene responses and enhanced levels of adaptive immune gene suppression compared to fish reared at higher density, resulting in significantly increased mortality rates. In addition, Saprolegnia infection substantially perturbs expression of circadian clock genes, with fish reared at low-density having higher levels of molecular clock dysregulation. CONCLUSIONS: Our results reveal the wide-scale impact of stocking density on transcriptional responses to infection and highlight the need to incorporate circadian biology into our understanding of disease dynamics in managed animals.

Molecular characterization and expression analysis of interleukin 15 (IL15) and interleukin-15 receptor subunit alpha (IL15Rα) in dojo loach (Misgurnus anguillicaudatus): Their salient roles during bacterial, parasitic and fungal infection.

Interleukin 15 (IL15) is a pleiotropic cytokine that participates in innate and adaptive immunity along with its receptor α-chain (IL15Rα). In order to investigate the potential roles of IL15 and IL15Rα in dojo loach (Misgurnus anguillicaudatus), we firstly cloned the cDNA sequence of Ma-IL15 and Ma-IL15Rα, which contain 1096bp and 1236bp and code proteins of 193 amino acids and 210 amino acids, respectively. A short signal peptide and Pfam IL15 domain were found in Ma-IL15, while a highly conserved sushi domain existed in Ma-IL15Rα. Ontogeny analysis indicated that significantly increased expression of Ma-IL15 and Ma- IL15Rα mRNA were detected in larvae from 1d to 7d post hatching, while relative high expression levels were detected in both systematic and mucosal immune-related tissues of adult dojo loach. Then three dojo loach infection models with F. columnare G4, I. multifiliis and Saprolegnia parasitica were constructed, which resulted in increased skin goblet cells and serious lesions in gill. Ma-IL15 and Ma-IL15Rα showed different expression patterns in different tissues during three infection models. Ma-IL15Rα mRNA was found to be more significantly elevated than Ma-IL15 after infection with F. columnare G4 in all examined tissues including kidney, spleen, gill and skin. I. multifiliis infection induced higher expression of Ma-IL15 in mucosal tissues including skin and gill, while it mainly increased Ma-IL15Rα expression in kidney. Moreover, our study firstly evaluated the influence of fungal infection on IL15 and IL15Rα expression in teleost, and it is interesting to find that both Ma-IL15 and Ma-IL15Rα expression showed consistent up-regulation after Saprolegnia parasitica infection compared to two other infection models. Therefore, our results suggest that Ma-IL15 and Ma-IL15Rα possess important defensive roles in systematic and mucosal tissues of dojo loach during bacterial, fungal and parasitic infection.

Cell entry of a host-targeting protein of oomycetes requires gp96.

The animal-pathogenic oomycete Saprolegnia parasitica causes serious losses in aquaculture by infecting and killing freshwater fish. Like plant-pathogenic oomycetes, S. parasitica employs similar infection structures and secretes effector proteins that translocate into host cells to manipulate the host. Here, we show that the host-targeting protein SpHtp3 enters fish cells in a pathogen-independent manner. This uptake process is guided by a gp96-like receptor and can be inhibited by supramolecular tweezers. The C-terminus of SpHtp3 (containing the amino acid sequence YKARK), and not the N-terminal RxLR motif, is responsible for the uptake into host cells. Following translocation, SpHtp3 is released from vesicles into the cytoplasm by another host-targeting protein where it degrades nucleic acids. The effector translocation mechanism described here, is potentially also relevant for other pathogen-host interactions as gp96 is found in both animals and plants.

Half the story: Thermal effects on within-host infectious disease progression in a warming climate.

Immune defense is temperature dependent in cold-blooded vertebrates (CBVs) and thus directly impacted by global warming. We examined whether immunity and within-host infectious disease progression are altered in CBVs under realistic climate warming in a seasonal mid-latitude setting. Going further, we also examined how large thermal effects are in relation to the effects of other environmental variation in such a setting (critical to our ability to project infectious disease dynamics from thermal relationships alone). We employed the three-spined stickleback and three ecologically relevant parasite infections as a "wild" model. To generate a realistic climatic warming scenario we used naturalistic outdoors mesocosms with precise temperature control. We also conducted laboratory experiments to estimate thermal effects on immunity and within-host infectious disease progression under controlled conditions. As experimental readouts we measured disease progression for the parasites and expression in 14 immune-associated genes (providing insight into immunophenotypic responses). Our mesocosm experiment demonstrated significant perturbation due to modest warming (+2°C), altering the magnitude and phenology of disease. Our laboratory experiments demonstrated substantial thermal effects. Prevailing thermal effects were more important than lagged thermal effects and disease progression increased or decreased in severity with increasing temperature in an infection-specific way. Combining laboratory-determined thermal effects with our mesocosm data, we used inverse modeling to partition seasonal variation in Saprolegnia disease progression into a thermal effect and a latent immunocompetence effect (driven by nonthermal environmental variation and correlating with immune gene expression). The immunocompetence effect was large, accounting for at least as much variation in Saprolegnia disease as the thermal effect. This suggests that managers of CBV populations in variable environments may not be able to reliably project infectious disease risk from thermal data alone. Nevertheless, such projections would be improved by primarily considering prevailing thermal effects in the case of within-host disease and by incorporating validated measures of immunocompetence.

Muli bamboo (Melocanna baccifera) leaves ethanolic extract a non-toxic phyto-prophylactic against low pH stress and saprolegniasis in Labeo rohita fingerlings.

Bamboo (Melocanna baccifera) plant plays a significant role in traditional Asian medicine and it can be used as an alternative to various chemo-therapeutics or prophylactic agents used in aquaculture. In the present study, the 1st experiment was done for the preparation of extracts from bamboo leaves by using 90% alcohol as a solvent. In the 2nd experiment, toxicity study was carried on Labeo rohita fingerlings (average length of 10.3 ± 1.5 cm and weight 18.5 ± 1.5 g) using BLAL (Bamboo Leaf Alcoholic) extract and it did not show any mortalities in fish even at the dose of 20 g kg-1 body weight, which can be regarded as virtually non-toxic with minimal effect. The 3rd experiment was conducted to find out the effect of BLAL extract based isocaloric and isonitrogenous feed (doses: control- 0.0%, diet T1-0.01%, diet T2-0.1%, diet T3-1% BLAL extract kg-1 feed) on the haemato-immuno-biochemical parameters of L. rohita fingerlings (average length 15.7 ± 1.5 cm and weight 20.2 ± 1.5 g). Regular sampling was done (on 7th, 15th, 30th and 60th day of feeding) for different haematological, immunological and biochemical parameters. The study revealed that the best response occurred within 7 days of feeding with BLAL extract and long-term feeding have shown immunosuppressive condition in the fish. In the 4th experiment, fish were exposed to multiple stressors like low pH stress, and Saprolegnia parastica infection after feeding with BLAL extract (0.1%) based feed for 7 days. The study showed that the BLAL extract can make fish resistant to single stress however, not able to reduce the fish mortality under multiple stresses (S. parastica infection under low pH). Thus, it can be concluded that the BLAL has the potentiality to be used as a non-toxic phyto-prophylactic which can induce non-specific immune response, reduces the low pH stress responses and increases the resistance against saprolegniasis under neutral pH condition in rohu fingerlings.

In vitro modulation of Drimys winteri bark extract and the active compound polygodial on Salmo salar immune genes after exposure to Saprolegnia parasitica.

The rapid development of the aquaculture industry has global concerns with health management and control strategies to prevent and/or treat diseases and increase sustainability standards. Saprolegniosis is a disease caused by Saprolegnia parasitica, and is characterized by promoting an immunosuppression in the host. This study evaluated in vitro the extract and one active compound (polygodial) of Drimys winteri, a Chilean medicinal tree as a potential early immunostimulatory aid in Saprolegniosis control. Atlantic salmon (Salmo salar) head kidney cells (ASK-1) were incubated with both extract and pure polygodial before exposure to S. parasitica mycelium, and the expression of the immune-related genes interleukin 1ß (IL-1ß), interferon α (IFNα), and major histocompatibility complex II (MHCII) was evaluated. Both evidenced immunomodulatory capacities by increasing gene expressions. This immunomodulation related to a mitigatory action counteracting the immunosuppressing effects of S. parasitica. Despite that most immune-related genes were up-regulated, the down-regulation of MHCII, characteristic of S. parasitica infection, was lessened by pre-incubation with the compounds. This study provides the first insight on the potential of D. winteri bark extract as a possible immunomodulatory and defensive strategy against this oomycete infection in fish.

Feeding pyridoxine prevents Saprolegnia parasitica infection in fish Labeo rohita.

A 60-day experiment was carried out to delineate the role of dietary pyridoxine (DP) in Labeo rohita fingerlings in modulating immunity and prevention of fungal infection. Two hundred and seventy fingerlings were randomly distributed into three treatments in triplicates. Three iso-caloric and iso-nitrogenous purified diets were prepared with graded levels of pyridoxine. Three experimental groups were C (0.0% DP), T1 (0.01% DP) and T2 (0.02% DP). The role of dietary pyridoxine in modulating immunity and prevention of fungal infection was assessed by haemato-immunological parameters like erythrocyte counts (EC), leucocyte counts (LC), haemoglobulin (Hb), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), nitro-blue tetrazolium (NBT), phagocytic activity, albumin, globulin, total plasma protein, albumin/globulin and by challenge study with Saprolegnia parasitica, where relative percentage survival (RPS) were recorded. Hb, PCV, MCV, MCH, NBT, total plasma protein, albumin, globulin contents, lysozyme and phagocytic activity was significantly (P < 0.05) higher in DP fed group. Significantly (P < 0.05) higher RPS was recorded from T2 group fed with 0.02% DP for 45 days. Hence, DP has the capacity to stimulate nonspecific immunity and increase resistance to S. parasitica infection in L. rohita fingerlings.

The effect of Ochratoxin A on antimicrobial polypeptide expression and resistance to water mold infection in channel catfish (Ictalurus punctatus).

Mycotoxin contamination of agricultural commodities poses a serious risk to animal health, including aquaculture species. Ochratoxin A (OA) is the most immunotoxic ochratoxin, yet little is known about its effect on immune function in fish. Antimicrobial polypeptides (AMPPs) are one of the most potent, innate, host defense factors, yet very little is known about what types of chronic stressors affect their expression. Among the most prevalent and potent AMPPs in fish are histone-like proteins (HLPs). In this study, fish were fed 2, 4, or 8 mg OA/kg diet. Skin antibacterial activity and HLP-1 levels were measured on Days 0, 28 and 56. Feeding 2, 4 or 8 mg OA/kg diet resulted in significant growth depression, but higher levels (4 or 8 mg OA/kg diet) resulted in lowering feed intake (FI) and impaired feed conversion ratio. In addition, feeding 8 mg OA/kg diet increased susceptibility to experimental water mold (Saprolegnia) challenge, suggesting that OA toxicity might contribute to some saprolegnosis outbreaks. However, there were no changes in AMPP expression in any treatment group. Our data suggests that the increased disease susceptibility of channel catfish due to OA is probably due to mechanisms other than a direct effect on antimicrobial polypeptide expression.

A thicker chorion gives ova of Atlantic salmon (Salmo salar L.) the upper hand against Saprolegnia infections.

Since the ban of malachite green in the fish farming industry, finding alternative ways of controlling Saprolegnia infections has become of utmost importance. Much effort has been made to elucidate the mechanisms by which Saprolegnia invades fish eggs. Little is known about the defence mechanisms of the hosts, making some eggs more prone to infection than others. One clue might lie in the composition of the eggs. As the immune system in the embryos is not developed yet, the difference in infection levels could be explained by factors influenced by the mother herself, by either transferring passive immunity, influencing the physical aspects of the eggs or both. One of the physical aspects that could be influenced by the female is the chorion, the extracellular coat surrounding the fish egg, which is in fact the first major barrier to be overcome by Saprolegnia spp. Our results suggest that a thicker chorion in eggs from Atlantic salmon gives a better protection against Saprolegnia spp. In addition to the identification of differences in sensitivity of eggs in a fish farm set-up, we were able to confirm these results in a laboratory-controlled challenge experiment.

Emerging oomycete threats to plants and animals.

Oomycetes, or water moulds, are fungal-like organisms phylogenetically related to algae. They cause devastating diseases in both plants and animals. Here, we describe seven oomycete species that are emerging or re-emerging threats to agriculture, horticulture, aquaculture and natural ecosystems. They include the plant pathogens Phytophthora infestans, Phytophthora palmivora, Phytophthora ramorum, Plasmopara obducens, and the animal pathogens Aphanomyces invadans, Saprolegnia parasitica and Halioticida noduliformans For each species, we describe its pathology, importance and impact, discuss why it is an emerging threat and briefly review current research activities.This article is part of the themed issue 'Tackling emerging fungal threats to animal health, food security and ecosystem resilience'.

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).