Comparative proteomic analysis provides insight into the key proteins as potential targets underlying the effect of malachite green against Ichthyophthirius multifiliis.

Target identification is important for drug discovery. Unfortunately, no drug targets have been found in Ichthyophthirius multifiliis until now and further limited development of the novel drug for Ichthyophthiriasis. In this study, an iTRAQ-based quantitative proteomic analysis was used to find the target of malachite green (MG), exhibiting greater efficacy than the existing drugs, against I. multifiliis trophonts in situ. We also verified the proteomic results by RT-qPCR, TEM and cell apoptosis assay. Our results showed that major variations in protein abundance were found among many of the ribosome proteins, indicating ribosome might be a candidate target. Furthermore, GO and KEGG pathway analyses of differentially expressed proteins (DEPs) revealed that ribosome and PI3K-Akt signalling pathway were remarkably enriched. Taken together, the above DEPs were also verified by RT-qPCR and morphological observations. This study provides insights into the key proteins enriched in PI3K-Akt signal pathway and ribosome pathway as potential targets of MG killing I. multifiliis, which could be served as targets for other less toxic drugs and be tested as potential treatments for I. multifiliis.

Evaluation of salinomycin isolated from Streptomyces albus JSY-2 against the ciliate, Ichthyophthirius multifiliis.

The present study was undertaken to investigate the antiparasitic activity of extracellular products of Streptomyces albus. Bioactivity-guided isolation of chloroform extracts affording a compound showing potent activity. The structure of the compound was elucidated as salinomycin (SAL) by EI-MS, 1H NMR and 13C NMR. In vitro test showed that SAL has potent anti-parasitic efficacy against theronts of Ichthyophthirius multifiliis with 10 min, 1, 2, 3 and 4 h (effective concentration) EC50 (95% confidence intervals) of 2.12 (2.22-2.02), 1.93 (1.98-1.88), 1.42 (1.47-1.37), 1.35 (1.41-1.31) and 1.11 (1.21-1.01) mg L-1. In vitro antiparasitic assays revealed that SAL could be 100% effective against I. multifiliis encysted tomonts at a concentration of 8.0 mg L-1. In vivo test demonstrated that the number of I. multifiliis trophonts on Erythroculter ilishaeformis treated with SAL was markedly lower than that of control group at 10 days after exposed to theronts (P < 0.05). In the control group, 80% mortality was observed owing to heavy I. multifiliis infection at 10 days. On the other hand, only 30.0% mortality was recorded in the group treated with 8.0 mg L-1 SAL. The median lethal dose (LD50) of SAL for E. ilishaeformis was 32.9 mg L-1.

Melaleuca alternifolia essential oil prevents bioenergetics dysfunction in spleen of silver catfish naturally infected with Ichthyophthirius multifiliis.

Some evidence has demonstrated that Ichthyophthirius multifiliis, etiologic agent of "white spot disease", causes severe bioenergetics dysfunction in the spleen of naturally infected silver catfish (Rhamdia quelen), which contributes directly to disease pathogenesis. Recently, several studies have demonstrated the efficacy of Melaleuca alternifolia essential oil, popularly known as tea tree oil (TTO), in the treatment of freshwater fish naturally or experimentally infected with I. multifiliis. In this sense, the aim of this study was to evaluate whether TTO is capable of preventing or reducing splenic bioenergetics dysfunction in silver catfish naturally infected with I. multifiliis. Splenic cytosolic and mitochondrial creatine kinase (CK) and pyruvate kinase (PK) activities decreased in infected animals compared to uninfected animals, while adenylate kinase (AK) activity increased. Treatment with TTO was able to prevent the inhibition on splenic CK and PK activities but was not able to prevent the stimulation of AK activity. Based on this evidence, treatment with TTO prevents the impairment on energetic metabolism via improvement of enzymes belonging to the phosphotransfer network, such as CK and PK. In summary, this treatment can be considered an interesting approach to prevent the bioenergetics imbalance in spleen of silver catfish naturally infected with I. multifiliis.

The in vitro and in vivo effect of tannic acid on Ichthyophthirius multifiliis in zebrafish (Danio rerio) to treat ichthyophthiriasis.

The in vitro antiparasitic effect of polyphenol tannic acid (TA) on Ichthyophthirius multifiliis theronts and tomonts was evaluated. In vitro antiparasitic assays revealed that TA in a dose- and time-dependent pattern through the damage of parasite plasma membrane could be 100% effective against I. multifiliis theronts at concentrations of 8 and 11 ppm during all the exposure times (45-270 min). The tomonts proliferation was completely inhibited by penetrating TA (at least 15 ppm for 22-hr exposure) into encysted tomont across the cyst wall. However, 10 ppm TA could result in a ninefold decrease in the population of live tomonts compared to the control group (p < 0.05). Although at theront concentrations of over 6,000 per zebrafish (Danio rerio), a 100% prevalence of ichthyophthiriasis during a 5-day exposure was recorded, results of in vivo tests showed that the parasite that pretreated up to 10 ppm TA for 70 min had not any capability to infect the studied zebrafish population. The acute toxicity (96 hr-LC50 ) of TA for zebrafish was 19.51 ppm. Thus, TA can be considered as a natural therapeutant to safely and efficiently improve the health of aquatic systems by controlling ichthyophthiriasis.

Impact of Pseudomonas H6 surfactant on all external life cycle stages of the fish parasitic ciliate Ichthyophthirius multifiliis.

A bacterial biosurfactant isolated from Pseudomonas (strain H6) has previously been shown to have a lethal effect on the oomycete Saprolegnia diclina infecting fish eggs. The present work demonstrates that the same biosurfactant has a strong in vitro antiparasitic effect on the fish pathogenic ciliate Ichthyophthirius multifiliis. Three life cycle stages (the infective theront stage, the tomont and the tomocyst containing tomites) were all susceptible to the surfactant. Theronts were the most sensitive showing 100% mortality in as low concentrations as 10 and 13 µg/ml within 30 min. Tomonts were the most resistant but were killed in concentrations of 100 µg/ml. Tomocysts, which generally are considered resistant to chemical and medical treatment, due to the surrounding protective cyst wall, were also sensitive. The surfactant, in concentrations of 10 and 13 µg/ml, penetrated the cyst wall and killed the enclosed tomites within 60 min. Rainbow trout fingerlings exposed to the biosurfactant showed no adverse immediate or late signs following several hours incubation in concentrations effective for killing the parasite. This bacterial surfactant may be further developed for application as an antiparasitic control agent in aquaculture.

Antiprotozoal effects of metal nanoparticles against Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis is a widespread, ciliated protozoan ectoparasite of fish. In the present study, we investigated the effects of metal nanoparticles on the reproduction and infectivity of free-living stages of I. multifiliis. We determined that ~50% of theronts could be killed within 30 min of exposure to either 20 ng mL-1 gold, 10 ng mL-1 silver or 5 ng mL-1 zinc oxide nanoparticles. Silver and zinc oxide nanoparticles at concentration of 10 and 5 ng mL-1 killed 100 and 97% of theronts, respectively and inhibited reproduction of tomonts after 2 h exposure. Gold nanoparticles at 20 ng mL killed 80 and 78% of tomonts and theronts 2 h post exposure, respectively. In vivo exposure studies using rainbow trout (Oncoryhnchus mykiss) demonstrated that theronts, which survived zinc oxide nanoparticles exposure, showed reduced infectivity compared with control theronts. No mortalities were recorded in the fish groups cohabited with theronts exposed to either nanoparticles compared with 100% mortality in the control group. On the basis of the results obtained from this study, metal nanoparticles particularly silver nanoparticles hold the best promise for the development of effective antiprotozoal agents useful in the management of ichthyophthiriosis in aquaculture.

Combined effects of Chinese medicine feed and ginger extract bath on co-infection of Ichthyophthirius multifiliis and Dactylogyrus ctenopharyngodonid in grass carp.

Dactylogyrus ctenopharyngodonid and Ichthyophthirius multifiliis are two important ectoparasites of freshwater fish. Co-infection by the two parasites leads to high fish mortality and results in heavy economic losses. This study aimed to evaluate the efficacy of medicated feed and a ginger extract bath against D. ctenopharyngodonid and I. multifiliis on grass carp and investigate the hematological response of grass carp co-infected by the two parasites. These results demonstrated that red blood cell (RBC) and thrombocyte percentage among leucocytes significantly decreased after grass carp were co-infected by D. ctenopharyngodonid and I. multifiliis. The monocyte and neutrophil percentages significantly increased with the increment of parasite mean intensities, while the lymphocyte percentage decreased. The activities of serum acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM), and superoxide dismutase (SOD) significantly increased after co-infection. When grass carp treated with medicated feed containing 4% of Astragalus membranaceus, Allium sativum, Morus alba, and Glycyrrhiza uralensis, the activities of ACP, AKP, LZM, and SOD were significantly enhanced, and the mean intensities of D. ctenopharyngodonid and I. multifiliis were significantly decreased. When grass carp was treated with medicated feed and a 4-mg/L ginger extract bath, all parasites were eliminated during 28 days. The bath of ginger extract at a concentration of 4 mg/L kept a low mean intensity of I. multifiliis and D. ctenopharyngodonid, then the two parasites were eliminated by oral administration of the medicated feed with an immunostimulant (Chinese medicine compound).

Essential oils to control ichthyophthiriasis in pacu, Piaractus mesopotamicus (Holmberg): special emphasis on treatment with Melaleuca alternifolia.

In vitro effect of the Melaleuca alternifolia, Lavandula angustifolia and Mentha piperita essential oils (EOs) against Ichthyophthirius multifiliis and in vivo effect of M. alternifolia for treating ichthyophthiriasis in one of the most important South American fish, Piaractus mesopotamicus (Holmberg), were evaluated. The in vitro test consisted of three EOs, each at concentrations of 57 µL L(-1) , 114 µL L (-1) , 227 µL L(-1) and 455 µL L (-1) , which were assessed once an hour for 4 h in microtitre plates (96 wells). The in vitro results demonstrated that all tested EOs showed a cytotoxic effect against I. multifiliis compared to control groups (P < 0.05). The in vivo treatment for white spot disease was performed in a bath for 2 h day(-1) for 5 days using the M. alternifolia EO (50 µL L (-1) ). In this study, 53.33% of the fish severely infected by I. multifiliis survived after the treatment with M. alternifolia (50 µL L (-1) ) and the parasitological analysis has shown an efficacy of nearly 100% in the skin and gills, while all the fish in the control group died. Furthermore, the potential positive effect of M. alternifolia EO against two emergent opportunistic bacteria in South America Edwardsiella tarda and Citrobacter freundii was discussed.

Two in vitro methods for screening potential parasiticides against Ichthyophthirius multifiliis using Tetrahymena thermophila.

Ichthyophthirius multifiliis (Ich) is a ciliate parasite that infects many species of freshwater fishes worldwide and causes heavy economic losses in aquaculture. Currently, parasiticides for controlling this parasite are limited, and few pond-practical chemical therapies exist. Hence, the search for new parasiticides is urgently needed. One challenge confronting the screening of potential parasiticides is the difficulty in raising enough parasite for efficacy testing as Ich is an obligate parasite. This study used species of Tetrahymena, Ich-related and cultivable ciliate protozoa, to evaluate two in vitro methods to test parasiticides. Plate counting and MTS assays (CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay) were used to compare lethal concentrations or median lethal concentrations (LC50) of copper sulphate, formalin and malachite green between T. thermophila and Ich theronts or between T. thermophila and Ich tomonts. The parasiticides that killed T. thermophila have been demonstrated to kill theronts or tomonts. These in vitro methods using T. thermophila can be used to screen novel parasiticides against Ich.

Evaluation of medicated feeds with antiparasitical and immune-enhanced Chinese herbal medicines against Ichthyophthirius multifiliis in grass carp (Ctenopharyngodon idellus).

Since malachite green was banned for using in food fish due to its carcinogenic and teratogenic effects on human, the search of alternative drug to treat Ichthyophthirius multifiliis becomes urgent. This study aimed to (1) evaluate the ethanol extracts of medicinal plants Cynanchum atratum, Zingiber officinale, Cynanchum paniculatum, immunostimulant (A), and immunostimulant (B) for their efficacy against I. multifiliis, and (2) determine effects of medicated feeds with C. atratum, Z. officinale, C. paniculatum, and immunostimulant (A) to treat I. multifiliis in grass carp. The results in this study showed that the minimum concentrations of C. atratum, Z. officinale, and C. paniculatum extracts for killing all theronts were 16, 8, and 16 mg/L, respectively. In vivo experiments, fish fed with medicated feeds of C. atratum for 10 days, or Z. officinale for 3 days, or combination of three plants for 10 days resulted in a significant reduction in the I. multifiliis infective intensity on grass carp after theronts exposure. Grass carp fed with medicated feeds of immunostimulant (A) for 21 days showed no infection and 100 % of survival 15 days post theronts exposure. Therefore, immunostimulant (A) is a promising feed supplement to treated I. multifiliis with good antiparasitic efficacy.

Antiparasitic efficacy of Gracillin and Zingibernsis newsaponin from Costus speciosus (Koen ex. Retz) Sm. against Ichthyophthirius multifiliis.

The present study aims to evaluate the antiparasitic activity of active components from Costus speciosus against Ichthyophthirius multifiliis. Bioassay-guided fractionation was employed to identify active compounds from C. speciosus yielding 2 bioactive compounds: Gracillin and Zingibernsis newsaponin. In-vitro assays revealed that Gracillin and Zingibernsis newsaponin could be 100% effective against I. multifiliis at concentrations of 0.8 and 4.5 mg L(-1), with median effective concentration (EC50) values of 0.53 and 3.2 mg L(-1), respectively. All protomonts and encysted tomonts were killed when the concentrations of Gracillin and Zingibernsis newsaponin were 1.0 and 5.0 mg L(-1). In-vivo experiments demonstrated that fish treated with Gracillin and Zingibernsis newsaponin at concentrations of 1.0 and 5.0 mg L(-1) carried significantly fewer parasites than the control (P<0.05). Mortality of fish did not occur in the treatment group (Zingibernsis newsaponin at 5.0 mg L(-1)) during the trial, although 100% of untreated fish died. Acute toxicities (LD50) of Gracillin and Zingibernsis newsaponin for grass carp were 1.64 and 20.7 mg L(-1), respectively. These results provided evidence that the 2 compounds can be selected as lead compounds for the development of new drugs against I. multifiliis.

Evaluation of an anti-parasitic compound extracted from Streptomyces sp. HL-2-14 against fish parasite Ichthyophthirius multifiliis.

The present study was conducted to evaluate the anti-parasitic activity of a pure compound from Streptomyces sp. HL-2-14 against fish parasite Ichthyophthirius multifiliis, and elucidate its chemical structure. By electron ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectrum (1H NMR and 13C NMR), the compound was identified as amphotericin B (AmB). The in vitro trials revealed that AmB can effectively kill the theronts and tomonts of I. multifiliis with the median lethal concentration (LC50) of 0·8 mg L-1 at 30 min for the theronts and 4·3 mg L-1 at 2 h for the tomonts, respectively. AmB at 5 mg L-1 significantly reduced I. multifiliis infectivity prevalence and intensity on grass carp (Ctenopharyngodon idella), and consequently decreased fish mortality, from 100% in control group to 30% in treated group. The 72 h acute toxicity (LC50) of AmB on grass carp was 20·6 mg L-1, but fish mortality was occurred when exposure to 13·0 mg L-1. These results indicated that AmB was effective in the therapy of I. multifiliis infection, but the safety concentration margin is relatively narrow. Further efforts aiming to decrease the toxicity and improve the therapeutic profile remain to be needed.

Evaluation of nystatin isolated from Streptomyces griseus SDX-4 against the ciliate, Ichthyophthirius multifiliis.

The present study was conducted to evaluate the in vitro and in vivo antiparasitic efficacy of active compounds from the bacterial extracellular products of Streptomyces griseus SDX-4 against Ichthyophthirius multifiliis. Bioassay-guided fractionation and isolation of compounds with antiparasitic activity were performed on n-butanol extract of S. griseus yielding a pure bioactive compound, nystatin (Nys), identified by comparing spectral data (EI-MS, (1)H NMR, and (13)C NMR) with literature values. Results from in vitro antiparasitic assays revealed that Nys could be 100% effective against I. multifiliis theronts and encysted tomonts at the concentration of 6.0 mg L(-1), with the median effective concentration (EC50) values of 3.1 and 2.8 mg L(-1) for theronts and encysted tomonts (4 h), respectively. Results of in vivo test demonstrated that the number of I. multifiliis trophonts on the gold fish treated with Nys was markedly lower than the control group at 10 days after exposed to theronts (p < 0.05). In the control group, 85.7% mortality was observed owing to heavy I. multifiliis infection at 10 days after the exposure. On the other hand, only 23.8% mortality owing to parasite infection was recorded in the groups treated with the Nys (4.0 and 6.0 mg L(-1)). In addition, our results showed that the survival and reproduction of I. multifiliis tomont exited from the fish were significantly reduced after treated with the 6.0 mg L(-1) Nys. The median lethal dose (LD50) of Nys for goldfish was 16.8 mg L(-1). This study firstly demonstrated that Nys has potent antiparasitic efficacy against I. multifiliis, and it can be a good candidate drug for chemotherapy and control of I. multifiliis infections.

Anti-parasitic activities of specific bacterial extracellular products of Streptomyces griseus SDX-4 against Ichthyophthirius multifiliis.

The ciliate Ichthyophthirius multifiliis is one of the most pathogenic parasites of fish maintained in captivity. In this study, effects of bacterial extracellular products of Streptomyces griseus SDX-4 against I. multifiliis were determined. The fermentation liquor of S. griseus was extracted successively in a separating funnel with petroleum ether, ethyl acetate, and n-butanol. In vitro assays revealed that the n-butanol extracts (NBu-E) and ethyl acetate extracts (Eto-E) of S. griseus were observed to be more effective against theronts than the other extracts with median effective concentration (EC50) values of 0.86 and 12.5 mg L(-1), respectively, and significantly reduced the survival of the tomonts and the total number of theronts released by the tomonts (P<0.05). All encysted tomonts were killed when the concentration of NBu-E was 30.0 mg L(-1). Results of in vivo test demonstrated that the number of I. multifiliis trophonts on the grass carp treated with NBu-E was markedly lower compared to the control group at 11 days after exposed to theronts (P<0.05). In the control group, 100% mortality was observed owing to heavy I. multifiliis infection at 11 days after the exposure. On the other hand, only 9.5% mortality owing to parasite infection was recorded in the groups treated with the NBu-E (30 mg L(-1)). The median lethal dose (LD50) of NBu-E for grass carp was 152.4 mg L(-1). Our results indicate that n-butanol extract of S. griseus will be useful in aquaculture for controlling I. multifiliis infections.

Minimum effective concentrations of formalin and sodium percarbonate on the free-living stages of an Australian isolate of Ichthyophthirius multifiliis.

Ichthyophthirius multifiliis Fouquet, 1876, a ciliate protozoan, is a common cosmopolitan parasite of freshwater teleosts and is a recurring problem during the summer months on Australian rainbow trout Oncorhynchus mykiss (Walbaum, 1792) farms. Preventative strategies include increasing water flow and filtration, but when an infection is established, chemical intervention is often required. Formalin (FOR) has been traditionally used on Australian trout farms as a treatment for I. multifiliis. Treatment using sodium percarbonate (SPC) that releases hydrogen peroxide when dissolved is being implemented on a number of farms. To assess anecdotal reports of low efficacy we evaluated 1 h exposures of FOR and SPC at 12 °C and 17 °C in both hard and soft water against free-living stages of I. multifiliis. Each free-living stage were exposed to FOR and SPC in vitro; theronts were exposed to 8, 16, 32 or 64 mg/l SPC or FOR every 15 min, for a maximum of 6 h, and the number of live theronts at each time point was recorded. Prototomonts and tomocysts were exposed to 64, 128, 256 and 512 mg/l SPC and 16, 32, 64 and 128 mg/l FOR for 1 h, incubated, with the percentage viability and the number of theronts produced recorded. Theronts were more sensitive to treatment than tomonts, and prototomonts were more sensitive to treatment than tomocysts. FOR and SPC killed all theronts within 15 min at 64 mg/l at both temperatures. FOR was effective against all prototomonts at ≥64 mg/l at both temperatures and was effective against all tomocysts at 128 mg/l at 17 °C but did not achieve complete mortality in any doses tested at 12 °C. SPC was effective against prototomonts and tomocysts at 64 m/l at 17 °C but required ≥256 mg/l at 12 °C. These results can be used to aid development of specific treatment strategies for the management of I. multifiliis on Australian rainbow trout farms.

Antiprotozoal screening of traditional medicinal plants: evaluation of crude extract of Psoralea corylifolia against Ichthyophthirius multifiliis in goldfish.

Ichthyophthirius multifiliis (also called "ich") is an external protozoan parasite that may infest almost all freshwater fish species and caused significant economic damage to the aquaculture industry. Since the use of malachite green was banned, there have been relatively few effective alternative strategies for controlling I. multifiliis infections. The present study was designed to screen potential antiparasitic medicinal plants based on our previous studies, and comprehensively evaluate in vitro and in vivo anti-ich activity of selected plant extracts. The screening results showed that the methanol extract of Psoralea corylifolia had the highest activity against I. multifiliis theronts. In vivo theront trials demonstrated that 1.25 mg/L or more concentrations of P. corylifolia methanol extract caused 100 % mortality during the 4-h exposure period, and the subsequent in vitro trials indicated that the minimum concentration of P. corylifolia methanol extract that prevented the initial infestation was 2.50 mg/L. Protomonts and encysted tomonts surviving trials suggested that encysted tomonts were less susceptible to P. corylifolia methanol extract than protomonts, and the methanol extract of P. corylifolia at a concentration of 5.00 mg/L could kill 100 % of protomonts and 88.89 % of encysted tomonts. It was also observed that after 12-h exposure of protomonts or encysted tomonts to 2.50 mg/L of P. corylifolia methanol extract, the theronts emerged from encysted tomonts led to more infection level than the ones in the other groups. The results suggested that whether the protomonts finish encystment is crucial to the survival, reproduction, and theronts infectivity. In addition, our results showed that long duration (24 h) and high concentration (5.00 mg/L) significantly reduced the survival and reproduction of I. multifiliis tomont exited from the fish after in-bath treatment, and it is indicated that P. corylifolia methanol extract had a potential detrimental effect on I. multifiliis trophont in situ.

Susceptibility of three strains of blue catfish, Ictalurus furcatus (Valenciennes), to Ichthyophthirius multifiliis.

This study compared the susceptibility of three blue catfish strains (D&B, USDA 101 and USDA 102) to the parasite Ichthyophthirius multifiliis (Ich). In Trial I, a cohabitation study (all strains stocked communally) was conducted and fish were exposed to theronts at 0, 200, 1000, 5000 or 25 000 theronts fish(-1), respectively. All fish died when exposed to theronts at 5000 or 25 000 theronts fish(-1). When exposed to 1000 theronts fish(-1), USDA 102 strain of blue catfish showed significantly lower mortality (78.5%) compared to USDA 101 and D&B strains (92.7% and 100%). In Trial II, the same three strains of blue fish were evaluated for their susceptibility to Ich with strains challenged in separate tanks by adding Ich theronts at 0, 200 and 1000 theronts fish(-1), respectively. All D&B and USDA 101 blue catfish died; however, 42.3% of USDA 102 strain survived the infection when exposed to 1000 theronts per fish. The results indicate that there are differences among strains of blue catfish for susceptibility to Ich, and these differences will be useful in the development of improved catfish germplasm for commercial aquaculture.

In vitro assessment of the chemotherapeutic action of a specific hydrogen peroxide, peracetic, acetic, and peroctanoic acid-based formulation against the free-living stages of Ichthyophthirius multifiliis (Ciliophora).

Traditionally, malachite green administrated as in-bath treatment was the most effective and common strategy used in freshwater aquaculture systems to control infections of the ciliate protozoan parasite Ichthyophthirius multifiliis Fouquet, 1876. After the ban of malachite green in the USA and Europe to be used in fish for human consumption, there has been extensive research destined to find efficacious replacements. Recently, peracetic acid-based compounds have demonstrated a strong cytotoxic effect in vitro and in vivo against I. multifiliis. In the present study, we tested the efficacy of a hydrogen peroxide, peracetic, acetic and peroctanoic acid-based formulation (HPPAPA) to eliminate the free-living stages of I. multifiliis (tomonts, cysts and theronts). The results obtained showed that the administration of low doses (8, 12 or 15 mg/l) of a specific HPPAPA-based product during a short window of exposure (60 min) kills nearly all free-living stages of I. multifiliis (theronts, tomonts and cysts) within the window of treatment (∼100% mortality for all the stages; one-way ANOVA, P ≤ 0.001). Of note, even the lowest concentration of HPPAPA tested (8 mg/l) was able to disrupt normal cyst development and therefore theront release. The demonstrated in vitro efficacy of the peracetic acid-based product tested on the present study suggests its great potential to control I. multifiliis infections in commercial aquacultural systems.

Antiprotozoal activity of medicinal plants against Ichthyophthirius multifiliis in goldfish (Carassius auratus).

Ichthyophthiriasis is a widespread disease in aquaculture and causes mass mortalities of fish. The development of new antiprotozoal agents for the treatment of Ichthyophthirius multifiliis infections is of increasing interest. The aim of the present study was to investigate the efficacy of 30 medicinal plants against I. multifiliis. The results showed that the methanol extracts of Magnolia officinalis and Sophora alopecuroides displayed the highest antiprotozoal activity against theronts, with 4-h LC(50) values estimated to be 2.45 and 3.43 mg L(-1), respectively. Concentrations of 2.5, 5.0, 10.0, and 20.0 mg L(-1) of M. officinalis extracts resulted in tomont mortality of 9.7, 43.7, 91.3, and 100% at 20 h, respectively. From 40 to 320 mg L(-1) of S. alopecuroides extracts, tomont mortality increased from 29.7 to 100%. Antiprotozoal efficacy against settled tomonts (2 and 10 h) was also applied; the results indicated that encysted I. multifiliis tomonts were less susceptible to these plant extract treatments. In vivo experiments demonstrated that high concentrations of M. officinalis and S. alopecuroides extracts could kill tomonts, and M. officinalis significantly reduced its reproduction (P < 0.05). These results suggested that the methanol extracts of M. officinalis and S. alopecuroides have the potential to be used as an eco-friendly approach for the control of I. multifiliis.

Molecular characterisation and biological activity of an antiparasitic peptide from Sciaenops ocellatus and its immune response to Cryptocaryon irritans.

Cryptocaryon irritans, a holotrichous ciliate parasitic protozoan, can trigger marine white spot disease and cause substantial economic losses in mariculture. However, methods of preventing and curing the disease have negatively affect fish, human, other organisms, and the natural environment. The antiparasitic activity of some antimicrobial peptides (AMPs) has garnered extensive attention of scholars. In this study, we identified and characterised a novel antiparasitic peptide, named So-pis, from Sciaenops ocellatus. The sequence analysis, structural features, and tissue distribution suggested that So-pis is genetically related to the piscidins family. However, So-pis showed a relatively low overall conservation compared with other known piscidins. So-pis is abound in glycine residues (22.7 %) and it has a neutral isoelectric point, weak amphipathicity, relatively long α-helix, and high hydrophobicity. These key elements are responsible for its biological activity. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicated that So-pis is a typically gill-expressed peptide. The expression of So-pis in the gill, skin, spleen, and head kidney could be regulated during C. irritans infection, thereby implicating a role of So-pis in immune defence against C. irritans. The synthetic So-pis had limited or no antimicrobial activity against bacterial and yeasts but exhibited potent antiparasitic activity against C. irritans in vitro. The activity of synthetic So-pis against erythrocytes was less potent than its antiparasitic activity against C. irritans. These results indicated that So-pis might be one of the crucial defence cytokines against C. irritans in the red drum. Cumulatively, our data suggested that So-pis might be a potential candidate for developing a novel, effective, and safe therapeutic agent against marine white spot disease.