INTRODUCTION
Ichthyophthirius multifiliis is a common protozoan parasite of freshwater fish, causing ‘white spot disease’ or ‘ichthyophthiriasis’ in tropical and sub-tropical regions with high mortality in aquarium and cultured fish (Shinn et al. Reference Shinn, Picón-Camacho, Bron, Conway, Yoon, Guo and Taylor2012). Typical pathologic changes of fish infected with I. multifiliis are white spots on the gills and skin and increased mucus production (Yao et al. Reference Yao, Shen, Li, Xu, Hao, Pan, Wang and Yin2010). The gills are highly susceptible to infection, and the resulting gill damage contributes considerably to the lethal effects caused by the parasite (Yao et al. Reference Yao, Shen, Li, Xu, Hao, Pan, Wang and Yin2010). There are four key developmental stages within the life cycle of I. multifiliis: the sub-epithelial, parasitic trophont (1) which, when mature, exits the host as a free-swimming tomont (2). Tomonts quickly settle on an appropriate substrate, encyst (3) and then undergo binary fission to produce tomites. Released tomites rapidly differentiate into theronts (4) which become infective (Matthews, Reference Matthews2005; Picón-Camacho et al. Reference Picón-Camacho, Marcos-Lopez, Bron and Shinn2012). Both the theront and tomont stages are free living in the life cycle. Killing the parasite at these stages will stop the reproductive cycle and prevent the spread of the disease (Straus and Griffin, Reference Straus and Griffin2001).
The most effective treatment of I. multifiliis has been achieved by use of malachite green, and mixtures of malachite green and formalin by immersion (Ekanem et al. Reference Ekanem, Obiekezie, Kloas and Knopf2004). However, due to the carcinogenic and genotoxic potentials of malachite green, it has been prohibited for use in the production of consumer fish in most of countries of the world (Heinecke and Buchmann, Reference Heinecke and Buchmann2009). Other chemicals, including copper sulphate, acriflavine, potassium permanganate, chloramine-T, sodium percarbonate and toltrazuril have also been used as chemotherapeutants for chemotherapy of ichthiophthiriasis (Tieman and Goodwin, Reference Tieman and Goodwin2001; Buchmann et al. Reference Buchmann, Jensen and Kruse2003; Ekanem et al. Reference Ekanem, Obiekezie, Kloas and Knopf2004). These chemicals aimed at interrupting the life cycle by killing the free-swimming stages of the parasite and achieved varying levels of success. Unfortunately, the continuous and long-term reliance on the limited kinds of chemicals has led to the development of drug resistance and causes environmental problems (Ling et al. Reference Ling, Wang, Liu, Li, Ye and Gong2010, Reference Ling, Wang, Wang and Gong2011, Reference Ling, Wang, Lu, Wang, Lui and Gong2012). There is, therefore, an urgent need to discover other effective and safe alternative anti-parasitic agents for the therapy of I. multifiliis infection.
Natural origin materials might be a reliable source for the discovery of novel and potential anti-parasitic agents (Li et al. Reference Li, Wan, Wang, Zhao and Wen2013). In recent years, increased research efforts have been focused on the utilization of natural origin components from plants or microorganisms to treat parasitic diseases in fish (Ekanem et al. Reference Ekanem, Obiekezie, Kloas and Knopf2004; Chu et al. Reference Chu, Zhang and Luo2010; Yao et al. Reference Yao, Shen, Li, Xu, Hao, Pan, Wang and Yin2010, Reference Yao, Zhou, Li, Yin, Ru, Pan, Hao, Xu and Shen2011; Zhang et al. Reference Zhang, Xu and Klesius2013). Yao et al. (Reference Yao, Shen, Li, Xu, Hao, Pan, Wang and Yin2010, Reference Yao, Zhou, Li, Yin, Ru, Pan, Hao, Xu and Shen2011) demonstrated the anti-I. multifiliis potential of some natural origin compounds such as sanguinarine, dihydrosanguinarine and dihydrochelerythrine. In another previous study, it was reported that n-butanol extracts of extracellular products from Streptomyces griseus SDX-4 were of high efficacy in controlling I. multifiliis infections and would be useful in aquaculture (Yao et al. Reference Yao, Yin, Li, Li, Xu, Ai and Shen2014). However, the active components responsible for the bioactivity had not been isolated in that study. We have previously demonstrated that a streptomycete strain (Streptomyces sp. HL-2-14) from East China Sea in Shanghai city was capable of producing anti-I. multifiliis components, and a pure compound was isolated from its fermentation products (unpublished data). The present study was conducted aiming to evaluate the anti-I. multifiliis efficacy of the isolated pure compound through in vitro and in vivo treatment trials, and elucidate its chemical structure by spectrum analyses.
MATERIALS AND METHODS
Uninfected fish
Healthy and uninfected grass carp (Ctenopharyngodon idella) with mean weight of 28·5 ± 2·1 g were obtained from a local aquaculture farm and acclimatized in a 1500-L glass aquarium under laboratory conditions (22·0 ± 1 °C, pH 7·1 ± 0·5, dissolved oxygen 6·1–7·2 mg L−1). The aquarium was fitted with an outside biological filter containing pea gravel. The skin surface and gills of 10 randomly sampled fish were examined under a microscope to confirm that fish were not infected with gill parasites or skin parasites. They were fed twice (9:00 and 17:00) per day at 3% body weight. All fish were acclimatized to laboratory conditions for 7 days before the experiment.
Parasites and preparation of theronts and tomonts
A strain of I. multifiliis was isolated from grass carp originating from a local fish pond. Laboratory populations were set up and the parasites were propagated on naive grass carp. The procedure was performed according to Heinecke and Buchmann, (Reference Heinecke and Buchmann2009). For production of theronts and tomonts a heavily infected fish was killed by an overdose anaesthetic (MS-222, Hunan Tiancheng Co. Ltd, China). The fish was placed in a 3 L aquarium containing 2 L municipal water and left in the water for 2–3 h (22–23 °C) allowing trophonts to escape from the epidermis and obtain the tomont stage. The tomonts were harvested from the water during these time intervals using a 200 μl pipette. For theront production, the sacrificed fish was removed after the respective time period and the aquarium was left for 24 h (22–23 °C) for the tomonts to encyst and theronts to develop. A high concentration of theronts suspension was produced by 2 min centrifugating at 1000 rpm. The concentrations of tomonts and theronts were determined by pipetting several microliter droplets of the suspension onto a glass slide and counting the organisms under an inverted microscope with ×40 magnification (Olympus IX71, Japan); the mean count in 10 droplets was extrapolated to determine the final concentration.
Bacteria and isolation of the pure compound
Extracts of fermentation products
Streptomyces sp. strain HL-2-14 (isolated from the soil samples in East China Sea) was inoculated in nutrient broth (NB) for 7 days on a reciprocal shaker water bath at 30 ± 1 °C. The combined fermentation liquor (300 L) was filtered, and the supernatant was separated by centrifuging at 3000 rpm for 15 min and then concentrated to dryness (344·8 g) by a vacuum freeze dryer (chemical-free, RiHong Co. Ltd. Changzhou city, China). The dry powder was suspended in distilled water and then was extracted successively in a separating funnel with petroleum ether, ethyl acetate and methanol. Each extract and the remaining aqueous part after solvent extraction were then evaporated to dryness under reduced pressure to give petroleum ether extract (PEE, 20·8 g), ethyl acetate extract (EAE, 61·4 g), methanol extract (ME, 240·5 g) and remaining aqueous extract (AE, 123·1 g). For the investigation of anti-parasitic efficacy, the dried extracts were tested against theronts in vitro.
Isolation procedure for pure compound
The ME showing the highest anti-parasitic efficacy against I. multifiliis theronts was subjected to further isolation according to the procedure reported previously (Wu et al. Reference Wu, Liu, Zhou, Wang, Xia and Liu2012) with a slight modification. Briefly, parts of the ME (200 g) were put through a silica gelcolumn (silica gel: 100–200 mesh) and eluted with solvent mixture of chloroform–methanol (1:0, 19:1, 9:1, 5:1, 2:1, 1:1, 1:2, 1:10 and 0:1, v⁄v), affording 641 fractions (300 mL each). Thin layer chromatography (TLC) analysis was performed on silica gel using the same solvent system as the mobile phase. Spots on TLC were visualized under ultraviolet (UV) light (254 and 365 nm) or by spraying the plates with ethanol–sulphuric acid reagent, and fractions showing similar chromatograms were combined eventually yielding seven new fractions A–G (Fr. A, 1–34; Fr. B, 35–96; Fr. C, 97–158; Fr. D, 159–281; Fr. E, 282–314; Fr. F, 315–501; Fr. G, 502–641). All new fractions were allowed to evaporate under vacuum until they got completely dry. A yellow crystal power (514·2 mg) was obtained from Fr. E by successive recrystallization process.
Anti-parasitic bioassays
For the measurement of anti-parasitic efficacy, in vitro trials against I. multifiliis theronts and tomonts along with in vivo trials against I. multifiliis infection were conducted. All trials were performed according to Yao et al. (Reference Yao, Yin, Li, Li, Xu, Ai and Shen2014). The extracts and the pure compound isolated from strain HL-2-14 were dissolved in dimethyl sulphoxide (DMSO) to get 1·0 g mL−1 (sample/solvent) of stocking solutions. Test solutions were obtained by diluting the stocking solutions with aerated and sterile groundwater.
In vitro trials against theronts
Trials were performed in 24-well tissue culture plates (Becton Dickinson Labware, NJ, USA). A total of 100 theronts were placed into each well of the plates in which filled with 2 mL different concentrations of test samples. The test concentrations were set as 30·0 and 50·0 mg L−1 for the extracts and 0·2, 0·4, 0·8, 1·6, 3·2, 6·4 and 12·8 mg L−1 for the pure compound. Anti-parasitic efficacy was determined by microscopic examination of each well at 30 min after treatment. Mortality was considered as the theronts with the absence of motility and integrity. A negative control was included using aerated groundwater containing the same amount of DMSO as the maximum concentration test group (<0·5%). All treatments and control groups were conducted with three replicates.
In vitro trials against tomonts
Fifty tomonts were placed into each well of a 6-well tissue culture plate (Becton Dickinson Labware, NJ, USA). Five milliliter of pure compound solutions at concentrations of 1·6, 3·1, 6·3, 12·5 and 25·0 mg L−1 were added to each well, respectively. A negative control was included using aerated groundwater containing the same amount of DMSO as the maximum concentration test group (<0·5%). The plates were incubated at 22 ± 0·5 °C. At 2 h after exposure, all solutions in each well were replaced by blank aerated groundwater (containing no test compound). The trial was allowed to stop until the parasites in the controls reached the theront stage. Mortality of theronts in each well (the parasites with the absence of internal cell motility or abnormal cell division and the ones cannot produce the theronts were considered dead) were recorded under microscope view. All treatments and control groups were conducted with three replicates.
In vivo trials against I. multifiliis infection
Two-hundred uninfected grass carp were transferred to 1000-L tanks for preparation of I. multifiliis infection. The tanks were aerated and supplied with groundwater. The temperature was maintained at 22 ± 0·5 °C using a heating device. Approximately 12 000 theronts/fish were put into the 1000-L tanks and incubated for 6 h with gentle aeration to promote infection. After the incubation, the fish were transferred to 50-L tanks with 20 infected fish per tank. Each tank was added with corresponding concentrations of the pure compound up to 1·3, 2·5 and 5·0 mg L−1, respectively. Control tank was also involved with no test sample. Triplicated tanks were used in each concentration and air stones were equipped in there. Daily observation was recorded for fish survival, and the number of trophonts on the fins and skin of fish was scored on day 3 after exposure. The trials ended at day 7.
Toxicity tests
Acute toxicity of the pure compound against the host (grass carp) was tested in 20-L glass tanks, each containing 10 L of test solution and 10 healthy grass carp. Dilutions were prepared from the stock solution as the following concentrations: 10·0, 13·0, 16·0, 19·0, 21·0 and 24·0 mg L−1. The tests were conducted in triplicate, as well as controls (under the same test conditions with no chemicals). Throughout the tests, fish were not fed, and the water quality was in consistence with those in acclimatization period. Fish mortality in the treatment and control groups were recorded after 72 h of exposure; and then median lethal concentration (LC50) was calculated.
Identification of the pure compound
Structure elucidation of the pure compound was performed using electrospray ionization mass spectrometry (ESI-MS; VG Co. Manchester, UK), nuclear magnetic resonance hydrogen spectrum (1H NMR) and nuclear magnetic resonance carbon spectrum (13C NMR) (Bruker, Madison, WI, USA).
Data analysis
The data in this study were analysed with SPSS version 16·0, and presented as mean ± standard deviation (s.d.). The homogeneity of the replicates of the samples was checked by the Mann–Whitney U test. Student–Newman–Keuls test was utilized for multiple comparisons. Probabilities of 0·05 or less were considered statistically significant. The median lethal concentration (LC50) with 95% confidence intervals (CI) was determined using the probit procedure.
RESULTS
Anti-theront efficacy of solvent extracts of strain HL-2-14
The results of anti-parasitic efficacies against I. multifiliis theronts of different solvent extracts (PEE, EAE, ME and AE) were depicted in Fig. 1, in which indicated ME is the extract having strongest anti-parasitic efficacy. At extract concentration of 50·0 mg L−1, 92·2% of theronts were dead after treated by ME for 30 min, while at the same concentration the theront mortality was 37·8, 32·2 and 58·9% for PEE, EAE and AE, respectively. At the concentration of 30·0 mg L−1, exposure to ME for 30 min resulted in I. multifiliis theront mortality by 76·7%. The solvent (DMSO) acted as a control showed no anti-parasitic activity when treated at the highest concentration.
Anti-theront efficacy of PEE, EAE, ME and AE of Streptomyces sp. HL-2-14 fermentation products against I. multifiliis at 30 min. Error bars denoted s.d. of 3 replicates. Statistically significant differences within a test concentration were indicated by the different letters (P < 0·05).
Anti-theront efficacy of the compound
In vitro trials showed that the pure compound was high in anti-parasitic efficacy against I. multifiliis theronts (Fig. 2). At the concentrations of 0·2, 0·4, 0·8 and 1·6 mg L−1, the treatment with the pure compound for 30 min led to a theront mortality of 20·1, 34·9, 54·9 and 85·4%, respectively. At the concentrations of 3·2, 6·4 and 12·8 mg L−1, the theronts were 100% dead. The calculated LC50 against the theronts was 0·8 mg L−1, with 95% CI of 0·69–0·81 mg L−1.
Anti-theront efficacy of the isolated compound from Streptomyces sp. HL-2-14 fermentation products against I. multifiliis at 30 min. Error bars denoted s.d. of 3 replicates. Statistically significant differences were indicated by the different letters (P < 0·05).
Anti-tomont efficacy of the compound
Figure 3 showed that the pure compound has a concentration-dependent effect on the survival of tomonts. When treated with the pure compound for 2 h, the tomonts was found 100% dead at 25 mg L−1. At the concentrations of 1·6, 3·1, 6·3, 12·5 mg L−1, the mortality was 29·0, 46·2 65·9 and 87·6%, respectively. The calculated LC50 was 4·3 mg L−1, with 95% CI of 3·74–4·77 mg L−1.
Anti-tomont efficacy of the isolated compound from Streptomyces sp. HL-2-14 fermentation products against I. multifiliis after 2 h exposure. Error bars denoted s.d. of 3 replicates. Statistically significant differences were indicated by the different letters (P < 0·05).
In vivo protecting efficacy of the compound
Supplementary Table S1 showed that the treatment with the pure compound significantly decreased the prevalence and the burden of I. multifiliis on grass carp, as compared to the control. At the control group, 100% of fish was infected with I. multifiliis at day 3 post-exposure, the average numbers of trophonts on skin and fins were 341·0 individuals, the fish were 100% dead at day 7. At the highest concentration group (5·0 mg L−1), the prevalence rate of I. multifiliis on the fish was significantly reduced to 46·7%, the average numbers of trophonts was 101·0 individuals, only 30·0% fish were dead at the end of the tests.
Acute toxicity
The acute toxicity tests revealed that the pure compound did not exhibit a lethal effect on the fish when exposure at 10·0 mg L−1 for 72 h (Fig. 4). Mortality of fish occurred when the concentration reached up to 13·0 mg L−1. At the highest concentration (24·0 mg L−1) for 72 h, 70% of fish were dead. The calculated 72-h LC50 was 20·6 mg L−1, with the 95% CI of 19·5–22·0 mg L−1.
Acute toxicity of the isolated compound from Streptomyces sp. HL-2-14 fermentation products against grass carp (C. idella) after 72 h exposure. Fish mortality was not occurred in 10·0 mg L−1 group. Error bars denoted standard deviation (s.d.) of 3 replicates. Statistically significant differences were indicated by the different letters (P < 0·05).
Structure identification
The chemical structure confirmations of the pure compound were accomplished by comparing its ESI-MS, 1H NMR and 13C NMR data with the literature values (Vandeputte et al. Reference Vandeputte, Wachtel and Stiller1955; Mechlinski et al. Reference Mechlinski, Schaffner, Gains and Avitabile1970; Aszalos et al. Reference Aszalos, Bax, Burlinson, Roller and Mcneal1985). The compound was identified as a known chemical, amphotericin B (hereafter abbreviated AmB), with the molecular formula of C47H73NO17. The chemical structure was shown in Fig. 5.
Chemical structure of AmB.
DISCUSSION
The in vitro assay may be an excellent tool for the screening of the anti-parasitic activity of potential drugs (extracts or compounds) (Ling et al. Reference Ling, Lu, Tu, Yi, Huang, Zhang and Wang2013). For the I. multifiliis, the theronts are the infective stage in their life cycle, it is important to kill theronts in order to control ichthyophthiriasis (Buchmann et al. Reference Buchmann, Sigh, Nielsen and Dalgaard2001). Therefore, in vitro anti-theront trials (theront mortality and survival at various time intervals as parameters) were usually used in evaluation of anti-parasitic activity against I. multifiliis in recent years (Shinn et al. Reference Shinn, Picón-Camacho, Bron, Conway, Yoon, Guo and Taylor2012; Ling et al. Reference Ling, Lu, Tu, Yi, Huang, Zhang and Wang2013; Zhang et al. Reference Zhang, Xu and Klesius2013). For example, Ling et al. (Reference Ling, Lu, Tu, Yi, Huang, Zhang and Wang2013) conducted an antiprotozoal screening of traditional medicinal plants against I. multifiliis in goldfish based on the in vitro anti-theront trials and reported that the crude extract of Psoralea corylifolia had the highest activity. Zhang et al. (Reference Zhang, Xu and Klesius2013) isolated an anti-I. multifiliis compound from ethanol extract of Galla chinensis according to the results of anti-theront trials. In the present study, the in vitro anti-theront trials were also used to investigate which solvent extract showing the strongest parasiticidal effect. The obtained results showed that the ME was the extract showing strongest parasiticidal effect, and was selected for further purification through column chromatography; eventually, a known chemical, i.e. AmB, was obtained.
To the best of our knowledge, this is first time that reports parasiticidal effect of AmB against fish parasites, although Yamasaki et al. (Reference Yamasaki, Harada, Tamura, Lim, Ohsuga, Yokoyama, Morishita, Nakamura, Ohta and Takiguchi2014) has reported that AmB exhibited in vitro and in vivo activities against a causative pathogen of canine babesiosis, Babesia gibsoni. The present results revealed that AmB killed all theronts of I. multifiliis at the concentration of 3·2 mg L−1 when exposure for 30 min. The calculated 30-min LC50 against the theronts was 0·8 mg L−1, with 95% CI of 0·69–0·81 mg L−1. These results suggest that the efficacy of AmB in the treatment of I. multifiliis theronts is a substantial improvement over many of the chemicals currently used or tested in an attempt to control this parasite (Shinn et al. Reference Shinn, Picón-Camacho, Bron, Conway, Yoon, Guo and Taylor2012; Zhang et al. Reference Zhang, Xu and Klesius2013). Shinn et al. (Reference Shinn, Picón-Camacho, Bron, Conway, Yoon, Guo and Taylor2012) tested the anti-I. multifiliis theronts efficacy of bronopol and reported that bronopol exposure at 100·0 mg L−1 for 30 min only killed the theronts by 52·7 ± 21·7%. Shinn et al. (Reference Shinn, Picón-Camacho, Bron, Conway, Yoon, Guo and Taylor2012) also demonstrated that formaldehyde applied at 200·0 mg L−1 for 1 h which kills 40% of theronts, hydrogen peroxide applied at 200·0 mg L−1 for 1 h which kills 15%; Virkon S at 10·0 mg L−1 for 1 h which kills 50%; salt (NaCl) at 5·0 ppt for 24 h which kills 90%, and chloramine T at 20·0 mg L−1 for 15 min which kills 20% of theronts. Zhang et al. (Reference Zhang, Xu and Klesius2013) assessed the anti-parasitic efficacy of pentagalloylglucose from G. chinensis against I. multifiliis in channel catfish (Ictalurus punctatus), and revealed that the mean mortality duration for killing all theronts in vitro of pentagalloylglucose was 165·8 ± 5·2 min when applied at 5·0 mg L−1. In addition, in the present study, it was found that the encysted tomonts resisted AmB better than the free-living theronts. This phenomenon was also found in other previous I. multifiliis treatment trials with other chemical (Buchmann et al. Reference Buchmann, Jensen and Kruse2003; Zhang et al. Reference Zhang, Xu and Klesius2013) or plant extracts (Ling et al. Reference Ling, Wang, Lu, Wang, Lui and Gong2012; Yi et al. Reference Yi, Lu, Hu, Ling and Wang2012). The in vivo results showed that treatment of AmB significantly reduced the prevalence of ichthyophthiriasis and the number of trophonts on fins and skin of fish on day 3 (Supplementary Table S1). The mortality of fish was also significantly reduced on day 7, from 100% in control group to 30% in 5·0 mg L−1 group. These results suggest that AmB was able to eliminate I. multifiliis and continuously immersing with this compound for 3 days is suitable for controlling I. multifiliis infection.
The chemical of AmB was firstly isolated from Streptomyces nodosus more than half a century ago (Kamiński et al. Reference Kamiński, Arczewska, Pociecha, Górecka, Stępniewski and Gagoś2014). It exhibiting a broad antimicrobial spectrum and high fungicidal activity is currently the drug of choice in the treatment of severe fungal infections (Hargreaves et al. Reference Hargreaves, Nguyen and Ryan2006). Structurally, this antibiotic possesses a macrolide ring, containing an internal ester or lactone, a heptane chromophore, and a hydroxyl hydrophilic region. The head of the molecule consists of a carboxyl group and an amino sugar, the latter being attached to the macrolide ring through a glycosidic linkage (Larabia et al. Reference Larabia, Gulikb, Dedieub, Legranda, Barratta and Cheron2004). According to the general conviction, the action mechanism of the drug is based on the formation of membrane pores that considerably affect membrane barrier function of cells (Arczewska and Gagoś, Reference Arczewska and Gagoś2011). The molecules of AmB adopt a quasi-parallel orientation with their polar sides (the polyhydroxyl groups) facing the interior of such formed pores and the hydrophobic parts interacting with the ergosterol in fungal cell membranes (Arczewska and Gagoś, Reference Arczewska and Gagoś2011). However, AmB also exhibits undesirable toxicity to mammalian cells (Herec et al. Reference Herec, Islamov, Kuklin, Gagos and Gruszecki2007). The structure of AmB causes this chemical has a tendency of self-association then leading to the formation of molecular aggregate. The self-associated AmB can form pores in cholesterol-containing membranes, leading to toxicity towards animal cells (Larabia et al. Reference Larabia, Gulikb, Dedieub, Legranda, Barratta and Cheron2004). In the present study, the mechanistic studies of AmB against I. multifiliis have not been included. The action mechanism of this chemical killing the theronts and tomonts of I. multifiliis was speculated to consist in change of membrane cell permeability, but the exact mechanism remains to be further investigated. The acute toxicity tests showed that AmB was relatively toxic to the fish. Mortality of fish occurred when the concentration of AmB reached up to 13·0 mg L−1, while AmB at 5·0 mg L−1 reduced the prevalence rate of I. multifiliis upon the fish from 100% in control groups to 46·7% (Supplementary Table S1 and Fig. 4). The safe range of AmB concentration is relatively narrow. Efforts aimed to decrease the toxicity to fish and improve the therapeutic profile of the chemical remains to be needed. Chemical derivatization and complexing the chemical within lipid-based formulations (liposomes, complexes and emulsions) are worth to be tried (Hing et al. Reference Hing, Schaefer and Kobayashi2000; Larabia et al. Reference Larabia, Gulikb, Dedieub, Legranda, Barratta and Cheron2004).
To summarize, the present study evaluated the anti-I. multifiliis efficacy of different solvent extracts of Streptomyces sp. HL-2-14, and a known chemical AmB was isolated from the ME. AmB could effectively killed theronts and tomonts of I. multifiliis in water. Treatment with the compound significantly reduced I. multifiliis infectivity prevalence and intensity, and consequently decreased the mortality. However, this compound was relatively toxic to the host (fish). The safe margin of chemical concentration was relatively narrow. Further studies will be needed to decrease the toxicity to fish and improve the therapeutic profile, through chemical techniques such as chemical derivatization.
SUPPLEMENTARY MATERIAL
To view supplementary material for this article, please visit http://dx.doi.org/10.1017/S0031182015000116.
ACKNOWLEDGEMENTS
We thank the key laboratory of East China Sea and oceanic fishery resources exploitation and utilization for their technic assistance. This paper was financially supported by the Key Project in Science and Technology Agency of Jilin Province (20150204065NY).
