Chemical Analysis and In Vitro Bioactivity of Essential Oil of Laurelia sempervirens and Safrole Derivatives against Oomycete Fish Pathogens.

In this study, the essential oil (EO) from Laurelia sempervirens was analyzed by GC/MS and safrole (1) was identified as the major metabolite 1, was subjected to direct reactions on the oxygenated groups in the aromatic ring and in the side chain, and eight compounds (4 to 12) were obtained by the process. EO and compounds 4-12 were subjected to biological assays on 24 strains of the genus Saprolegnia, specifically of the species 12 S. parasitica and 12 S. australis. EO showed a significant effect against Saprolegnia strains. Compound 6 presents the highest activity against two resistant strains, with minimum inhibitory concentration (MIC) and minimum oomyceticidal concentration (MOC) values of 25 to 100 and 75 to 125 µg/mL, respectively. The results show that compound 6 exhibited superior activities compared to the commercial controls bronopol and azoxystrobin used to combat these pathogens.

Anti-oomycetes and immunostimulatory activity of natural plant extract compounds against Saprolegnia spp.: Molecular docking and in-vitro studies.

This study aimed to investigate the effectiveness of five natural plant extract compounds Curcumin (CUR); Eugenol (EUG), Cinnamaldehyde (CIN), Stigmasterol (ST) and Morin (MOR), on two species of Saprolegnia; Saprolegnia parasitica and S. australis. Selective compounds were screened for the minimum inhibitory concentration, first for anti-oomycetes activity and then mycelium growth inhibition, spore germination inhibition and colonisation test. Nitric oxide production and myeloperoxidase activity of the compounds were tested in head kidney leukocytes of rainbow trout, Oncorhynchus mykiss to assess the immunostimulatory potential. Molecular docking of effective compounds was carried out with effector proteins of S. parasitica to investigate the target binding sites. Among all, CUR could completely inhibit zoospore production and significantly (p ≤ .05) inhibit hyphal growth at 16 mg l-1 against S. parasitica and S. australis. CIN at the concentration of 50 mg l-1 completely inhibited hyphal growth of both Saprolegnia spp., although the zoospore production of S. parasitica and S. australis was reduced at 25 mg l-1 and 10 mg l-1. In the case of EUG, significant inhibition of the hyphal growth and germination of S. parasitica zoospores was observed at 50 mg l-1. ST and MOR did not show antioomycetes activity. The molecular docking results were consistent with in vitro studies, possibly due to the binding with the vital proteins (Plasma membrane ATPase, V-type proton ATPase, TKL protein kinase, Host targeting protein 1) of S. parasitica and ultimately inhibiting their activity. CUR and CIN showed increased nitric oxide production at the highest concentration of 250 and 256 mg l-1 but the value was not significant (p ≤ .05) with control. CUR showed significantly higher peroxidase activity (p ≤ .05) at a concentration of 256 mg l-1 though values were significantly similar with concentration from 16 to 128 mg l-1. The nitric oxide and total peroxidase activity of rainbow trout leukocytes in the case of CIN showed a significant difference only at 250 mg l-1 against the control. The results conclude that CUR, CIN showed the better anti-Saprolegnia activity and could be used as phyto-additives in aquaculture. Among all, the inclusion of CUR as phyto-additives will provide additional immunostimulatory activity.

Thyme essential oil loaded microspheres for fish fungal infection: microstructure, in vitro dynamic release and antifungal activity.

AIMS: Evaluate the effect of varying the droplet size of microspheres charged with thyme essential oil (TEO-MS) on their swelling (Sw), release rate (%RR) and in vitro antifungal activity against Saprolegnia sp. METHODS: TEO-MS obtained by ionic gelation were characterised through SEM microscopy and X-ray microtomography. Their Sw and RR% were evaluated at simulated fish-gastrointestinal conditions using gravimetric and spectrophotometric techniques. RESULTS: For all evaluated droplet sizes (p ≥ 0.05), TEO was heterogeneously distributed inside of the MS and TEO-MS experimented agglomeration and sphericity loss after the drying process. Under gastric conditions, the acid pH (2.9) limited the Sw (50-100%) of TEO-MS, generating a low RR% (14-18%). Contrary, the slightly alkaline intestinal pH (8.1) favoured the Sw (∼3.2 to 3.8 times) and therefore the RR% (42-63%). CONCLUSIONS: TEO-MS (5-100 mg/mL) presented antifungal capacity onto Saprolegnia sp. after the simulated fish digestion, being the small droplet size once the most effective.

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.

Efficacy and safety of boric acid as a preventive treatment against Saprolegnia infection in Nile tilapia (Oreochromis niloticus).

Saprolegniosis is a worldwide fungal-like infection affecting freshwater fishes and their eggs. Reports show high mortalities and subsequent economic losses annually from Saprolegnia infections. Most therapeutants against Saprolegnia spp. infections are inefficient and some have negative impact on the environment. In this study, we have investigated the ability of boric acid (BA) to prevent Saprolegnia infection in Nile tilapia (Oreochromis niloticus). BA inhibited radial growth of Saprolegnia hyphae in vitro. Complete in vitro growth inhibition was found at a concentration of ≥0.6 g/L. Inhibitory effects were also observed in vivo when Nile tilapia were experimentally challenged with Saprolegnia spores and followed over 10 days post challenge and under continuous exposure to different BA concentrations. No signs of saprolegniosis were observed in fish treated with BA at concentrations of 0.4 g/L and above. Comet assay revealed that BA has low toxicity in tilapia continuously exposed to concentrations of 0.2-0.6 g/L for 96 h. Additionally, no significant histomorphological changes were observed in BA-treated fish compared to non-treated controls. Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) enzyme levels indicated reduction in systemic tissue damage associated with Saprolegnia infection. This study demonstrates the potential of BA as a prophylactic measure against Saprolegnia infection in tilapia, and we recommend additional studies on environmental impact.

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.

In Vitro Activity of Essential Oils against Saprolegnia parasitica.

Saprolegnia spp. water molds severely impact fish health in aquaculture, fish farms and hobby fish tanks colonizing mature and immature stages of fishes, as well as eggs. Considering that there are no drugs licensed for treating and/or control the organism, efficient and environmental low-impact methods to control these oomycetes in aquaculture are needed. The aim of the present report was to evaluate the in vitro sensitivity of Saprolegnia parasitica to essential oils (EOs) from Citrus aurantium L., Citrus bergamia Risso et Poiteau, Citrus limon Burm. f., Citrus paradisi Macfad, Citrussinensis Osbeck, Cinnamomum zeylanicum Blume, Cymbopogon flexuosum (Nees ex Steud.) Watson, Foeniculum vulgare Mill., Illicium verum Hook.f., Litsea cubeba (Lour.) Pers., Origanum majorana L., Origanum vulgare L., Pelargonium graveolens L'Hér., Syzygium aromaticum Merr. & L.M.Perry, and Thymus vulgaris L., by microdilution test. The most effective EOs assayed were T. vulgaris and O. vulgare, followed by C. flexuosum, L. cubeba and C. bergamia. These EOs could be of interest for controlling Saprolegnia infections. Nevertheless, further safety studies are necessary to evaluate if these products could be dispersed in tank waters, or if their use should be limited to aquaculture supplies.

Synthesis of dihydroisorcordoin derivatives and their in vitro anti-oomycete activities.

A series of novel dihydrochalcone derivatives 2-7 were synthesized from dihydroisorcordoin 1 which was isolated from the aerial parts of Adesmia balsamica. The structures of all compounds were confirmed by 1H NMR, 13C NMR, and HRMS. Their anti-oomycete activity was evaluated in vitro against Saprolegnia parasitica and Saprolegnia diclina. Some of the newly synthesized compounds exhibited better anti-oomycete activities at low values compared with bronopol and fluconazole as positive controls. Among them, compound 7 exhibited strong activity, with minimum inhibitory concentration and minimum oomyceticidal concentration values of 75 and 100 µg/mL, respectively.

In vitro activity of chemicals and commercial products against Saprolegnia parasitica and Saprolegnia delica strains.

Oomycetes of the genus Saprolegnia are responsible for severe economic losses in freshwater aquaculture. Following the ban of malachite green in food fish production, the demand for new treatments pushes towards the selection of more safe and environment-friendly products. In the present work, in vitro activity of ten chemicals and three commercial products was tested on different strains of Saprolegnia, using malachite green as reference compound. The compounds were screened in agar and in water to assess the minimum inhibitory concentration (MIC) and the minimum lethal concentration (MLC), respectively. Two strains of Saprolegnia parasitica and one isolate of Saprolegnia delica were tested in triplicate per each concentration. Among tested chemicals, benzoic acid showed the lowest MIC (100 ppm) followed by acetic acid, iodoacetic acid and copper sulphate (250 ppm). Sodium percarbonate was not effective at any tested concentration. Among commercial products, Virkon™ S was effective in inhibiting the growth of the mycelium (MIC = MLC = 1,000 ppm). Actidrox® and Detarox® AP showed MIC = 5,000 and 1,000 ppm, respectively, while MLCs were 10-fold lower than MICs, possibly due to a higher activity of these products in water. Similarly, a higher effectiveness in water was observed also for iodoacetic acid.

Isolation of anti-Saprolegnia lignans from Magnolia officinalis and SAR evaluation of honokiol/magnolol analogs.

To control the fish fungal pathogen Saprolegnia, the effects of the petroleum ether extracts of Magnolia officinalis were evaluated by a rapeseed (Brassicanapus) microplate method in vitro. By loading on an open silica gel column and eluting with petroleum ether-ethyl acetate-methanol, honokiol (C18H18O2) and magnolol (C18H18O2) were isolated from Magnolia officinalis. Saprolegnia parasitica growth was inhibited significantly when honokiol concentration was >8.0 mg/L, and magnolol concentration was >9.0 mg/L, with EC50 values of 4.38 and 4.92 mg/L, respectively. Six honokiol and magnolol derivatives were designed, synthesized and evaluated for their anti-Saprolegnia activity. According to the results, double bond and hydroxyl played an important role in inhibiting Saprolegnia. Mechanistically, through the scanning electron microscope observation, honokiol and magnolol could cause the Saprolegnia parasitica mycelium tegumental damage including intensive wrinkles and nodular structures. Moreover, compared to traditional drugs kresoxim-methyl (LC50 = 0.66 mg/L) and azoxystrobin (LC50 = 2.71 mg/L), honokiol and magnolol showed a lower detrimental effect on zebrafish, with the LC50 values of 6.00 and 7.28 mg/L at 48 h, respectively. Overall, honokiol and magnolol were promising lead compounds for the development of commercial drugs anti-Saprolegnia.

Effect of miconazole nitrate on immunological response and its preventive efficacy in Labeo rohita fingerlings against oomycetes Saprolegnia parasitica.

This study evaluated the effect of sublethal doses of antifungal drug miconazole nitrate (MCZ) on immunological responses and its role as a prophylactic drug against S. parasitica in Labeo rohita fingerlings. Fish were fed with sublethal doses of MCZ, that is, T1-6.30 mgMCZ kgBW-1 , T2-12.61 mgMCZ kgBW-1 and T3-25.22 mgMCZ kgBW-1 , and sampling was done at different time intervals for 240 hr. Immunological parameters viz. lysozyme activity, oxygen radical production and plasma antiprotease activity showed significant enhancement (p < 0.05) in fish fed with T2 and T3 doses. Expression of immune-relevant genes such as TLR-22 and ß2-M showed significantly higher expression at 6 hr and 24 hr of sampling in both liver and head kidney. However, these genes showed a downregulation after 120 hr of sampling in both the tissues. Preventive efficacy study showed that single dose of MCZ provides protection against oomycetes up to the fourth day of infection. Significantly higher mortality was observed in control diet-fed fish as compared to fish fed with MCZ medicated diet. Thus, it can be concluded that the MCZ can act as a potent antifungal agent for preventing oomycetes infection as well as to enhance the immune response.

Structure-Activity Relationship of Dialkoxychalcones to Combat Fish Pathogen Saprolegnia australis.

To investigate the anti-Saprolegnia activities of chalconic compounds, nine dialkoxychalcones 2⁻10, along with their key building block 2',4'-dihydroxychalcone 1, were evaluated for their potential oomycide activities against Saprolegnia australis strains. The synthesis afforded a series of O-alkylated derivatives with typical chalcone skeletons. Compounds 4⁻10 were reported for the first time. Interestingly, analogue 8 with the new scaffold demonstrated remarkable in vitro growth-inhibitory activities against Saprolegnia strains, displaying greater anti-oomycete potency than the standard drugs used in the assay, namely fluconazole and bronopol. In contrast, a dramatic loss of activity was observed for O-alkylated derivatives 2, 3, 6, and 7. These findings have highlighted the therapeutic potential of the natural compound 1 scaffold to be exploitable as a drug lead with specific activity against various Saprolegnia strains.

Quellenin, a new anti-Saprolegnia compound isolated from the deep-sea fungus, Aspergillus sp. YK-76.

Saprolegnia parasitica, belonging to oomycetes, is one of virulent pathogen of fishes such as salmon and trout, and causes tremendous damage and losses in commercial aquacultures by saprolegniasis. Previously, malachite green, an effective medicine, had been used to control saprolegniasis. However, this drug has been banned around the world due to its mutagenicity. Therefore, novel anti-saprolegniasis compounds are urgently needed. As a new frontier to discover bioactive compounds, we focused on the deep-sea fungi for the isolation of anti-saprolegniasis compounds. In this paper, on the course of anti-saprolegniasis agents from 546 cultured broths of 91 deep-sea fungal strains, we report a new compound, named quellenin (1) together with three known compounds, diorcinol (2), violaceol-I (3) and violaceol-II (4), from deep-sea fungus Aspergillus sp. YK-76. This strain was isolated from an Osedax sp. annelid, commonly called bone-eating worm, collected at the São Paulo Ridge in off Brazil. Compounds 2, 3 and 4 showed anti-S. parasitica activity. Our results suggest that diorcinol and violaceol analogs and could be good lead candidates for the development of novel agents to prevent saprolegniasis.

Effects of fluconazole based medicated feed on haemato-immunological responses and resistance of Labeo rohita against Saprolegnia parasitica.

Fluconazole (FLZ) is a new azole antifungal drug having no earlier record of its utilization for the treatment of fish diseases. A 55-days experiment was carried out to delineate the role of FLZ based medicated feed on haemato-immunological responses and prevention of fungal infection in Labeo rohita fingerlings. Three hundred and sixty fingerlings were randomly distributed into four experimental groups in triplicates. Four iso-caloric and iso-nitrogenous purified medicated feeds were prepared with 0, 100, 200 and 300 mg FLZ 100 g-1 feed. Haemato-immunological parameters like erythrocyte counts (EC), haemoglobulin (Hb), leucocyte counts (LC), packed cell volume (PCV), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), nitro-blue tetrazolium (NBT), albumin, globulin, total plasma protein were studied. The present study revealed that the continuous feeding of FLZ based feed for 15-30 days significantly (P < 0.05) increases the erythropoiesis, heme-synthesis, as well as the leucocytosis in rohu. However, all the doses exhibited equal protection from the infectious S. parasitica after 45 days of continuous feeding.

Hemi-Synthesis and Anti-Oomycete Activity of Analogues of Isocordoin.

An efficient synthesis of a series of 4'-oxyalkyl-isocordoin analogues (2-8) is reported for the first time. Their structures were confirmed by ¹H-NMR, 13C-NMR, and HRMS. Their anti-oomycete activity was evaluated by mycelium and spores inhibition assay against two selected pathogenic oomycetes strains: Saprolegnia parasitica and Saprolegnia australis. The entire series of isocordoin derivatives (except compound 7) showed high inhibitory activity against these oomycete strains. Among them, compound 2 exhibited strong activity, with minimum inhibitory concentration (MIC) and minimum oomyceticidal concentration (MOC) values of 50 µg/mL and 75 µg/mL, respectively. The results showed that 4'-oxyalkylated analogues of isocordoin could be potential anti-oomycete agents.

A fluorescence-based assay for in vitro screening of Saprolegnia inhibitors.

The incidence of fish pathogenic oomycetes, Saprolegnia, has increased significantly in aquaculture since the ban of malachite green. For the efficient characterization of anti-Saprolegnia therapeutics, simple accurate methods are required. However, the current screening methods are limited by time, and none of them are confirming the viability of treated spores or hyphae. In this study, a modified fluorescence-based assay for the in vitro screening of Saprolegnia inhibitors has been developed. This method involves the use of FUN-1 viability dye combined with calcofluor white M2R, and is based on the formation of orange-red cylindrical intravacuolar structures (CIVS) in metabolically active spores, hyphae and biofilms. Heat-killed and bronopol-treated Saprolegnia spores, hyphae and biofilms exhibited diffuse bright green fluorescence which confirms complete loss of viability. For boric acid-treated spores, no germination was observed. However, tiny CIVS were observed in 50% of treated spores which indicated reduction in their viability. Our results proved that FUN-1 dye is an efficient tool to distinguish between live and dead Saprolegnia spores, hyphae and biofilms and to monitor the change in Saprolegnia viability during qualitative evaluation of potential anti-Saprolegnia compounds.

Synthesis and Evaluation of Novel Oxyalkylated Derivatives of 2',4'-Dihydroxychalcone as Anti-Oomycete Agents against Bronopol Resistant Strains of Saprolegnia sp.

A series of novel oxyalkylchalcones substituted with alkyl groups were designed and synthesized, and the antioomycete activity of the series was evaluated in vitro against Saprolegnia strains. All tested O-alkylchalcones were synthesized by means of nucleophilic substitution from the natural compound 2',4'-dihydroxychalcone (1) and the respective alkyl bromide. The natural chalcone (1) and 10 synthetic oxyalkylchalcones (2-11) were tested against Saprolegnia parasitica and Saprolegnia australis. Among synthetic analogs, 2-hydroxy,4-farnesyloxychalcone (11) showed the most potent activity against Saprolegnia sp., with MIC and MOC values of 125 µg/mL (similar to bronopol at 150 µg/mL) and 175 µg/mL, respectively; however, 2',4'-dihydroxychalcone (1) was the strongest and most active molecule, with MIC and MOC values of 6.25 µg/mL and 12.5 µg/mL.

Inhibition of dioscin on Saprolegnia in vitro.

As one of the most serious pathogens in the freshwater aquatic environment, Saprolegnia can induce a high mortality rate during the fish egg incubation period. This study investigated the anti-Saprolegnia activity of a total of 108 plants on Saprolegnia parasitica in vitro and Dioscorea collettii was selected for further studies. By loading on an open silica gel column and eluting with petroleum ether-ethyl acetate-methanol, dioscin (C45H72O16) was isolated from D. collettii. Saprolegnia parasitica growth was inhibited significantly when dioscin concentration was more than 2.0 mg L(-1). When compared with formalin and hydrogen peroxide, dioscin showed a higher inhibitory effect. As potential inhibition mechanisms, dioscin could cause the S. parasitica mycelium morphologic damage, dense folds, or disheveled protuberances observed by field emission scanning electron microscopy and the influx of Propidium iodide. The structural changes in the treated mycelium were indicative of an efficient anti-Saprolegnia activity of dioscin. The oxidative stress results showed that dioscin also accumulated reactive oxygen species excessively and increased total antioxidant and superoxide dismutase activity. These situations could render S. parasitica more vulnerable to oxidative damage. Additionally, when dioscin concentration was less than 2.0 mg L(-1), the survival rate of embryos was more than 70%. Therefore, the use of dioscin could be a viable way of preventing and controlling saprolegniasis.

Chemical Characterization and Anti-Oomycete Activity of Laureliopsis philippianna Essential Oils against Saprolegnia parasitica and S. australis.

Laureliopsis philippiana (Looser) R. Schodde (Monimiaceae) is a native tree widespread in the forest areas in the south of Chile and Argentina, known for its medicinal properties and excellent wood. The aim of this study was to evaluate the chemical composition of L. philippiana leaf and bark essential oils (EOs) using gas chromatography-mass spectrometry (GC-MS), and to quantify its anti-oomycete activity, specifically against Saprolegnia parasitica and S. australis. Only six components were identified in leaf EO, 96.92% of which are phenylpropanoids and 3.08% are terpenes. As for bark EO, 29 components were identified, representing 67.61% for phenylpropanoids and 32.39% for terpenes. Leaf EO was characterized mainly by safrole (96.92%) and ß-phellandrene (1.80%). Bark EO was characterized mainly by isosafrole (30.07%), safrole (24.41%), eucalyptol (13.89%), methyleugenol (7.12%), and eugenol (6.01%). Bark EO has the most promising anti-Saprolegnia activity, with a minimum inhibition concentration (MIC) value of 30.0 µg/mL against mycelia growth and a minimum fungicidal concentration (MFC) value of 50.0 µg/mL against spores; for leaf EO, the MIC and MFC values are 100 and 125 µg/mL, respectively. These findings demonstrate that bark EO has potential to be developed as a remedy for the control of Saprolegnia spp. in aquaculture.