Next Article in Journal
Inhibition Effects of Nippostrongylus brasiliensis and Its Derivatives against Atherosclerosis in ApoE-/- Mice through Anti-Inflammatory Response
Previous Article in Journal
The Prevalence of Pathogens among Ticks Collected from Livestock in Kazakhstan
Previous Article in Special Issue
Physicochemical Water Quality Influence on the Parasite Biodiversity in Juvenile Tilapia (Oreochromis spp.) Farmed at Valle Del Mezquital in the Central-Eastern Socioeconomic Region of Mexico
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Pathogens
Volume 11
Issue 10
10.3390/pathogens11101207
pathogens-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Brief Report

In Vitro Assessment of Berberine against Ichthyophthirius multifiliis in Goldfish

by
Ke Huang
1,2,
Guangran Hu
1,2,
Runqiu Wang
3,
Qingwen Zeng
1,2,
Wenxiang Li
1,
Hong Zou
1,
Shangong Wu
1,
Guitang Wang
1 and
Ming Li
1,*
1
State Key Laboratory of Freshwater Ecology and Biotechnology, and Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
2
University of Chinese Academy of Sciences, Beijing 100049, China
3
Hubei Key Laboratory of Three Gorges Project for Conservation of Fishes, Chinese Sturgeon Research Institute, China Three Gorges Corporation, Yichang 443100, China
*
Author to whom correspondence should be addressed.
Pathogens 2022, 11(10), 1207; https://doi.org/10.3390/pathogens11101207
Submission received: 25 September 2022 / Revised: 16 October 2022 / Accepted: 18 October 2022 / Published: 20 October 2022
(This article belongs to the Special Issue Parasites Harnessing the Aquaculture: More We Know, More Questions We Rise)
Browse Figures
Review Reports Versions Notes

Abstract

:
Ichthyophthirius multifiliis is a pathogenic ciliate parasite, which infects almost all freshwater teleost fish and leads to significant economic losses. The present study aimed to evaluate the acute toxicity of berberine to the free-living stages of I. multifiliis, that is, theronts and tomonts. Our results indicated that 99.30% of I. multifiliis theronts were killed by a concentration of 15 mg/L berberine during the 4 h exposure time, while berberine had no effect on protomonts. Nevertheless, berberine at a concentration of 5 mg/L could effectively reduce the release of theronts from tomonts treated for 4 h. Additionally, according to the transmission electron microscopy results, berberine at 15 mg/L could strongly change the shape of protomonts, destroy their organelles, and significantly decrease the number of ribosomes. The median lethal concentration (LC50) of berberine for goldfish at 96 h was 528.44 mg/L, which was almost 67 times the median effective concentration (EC50) of berberine for killing theronts (7.86 mg/L). The results demonstrated that berberine could be an effective and safe potential parasiticide for killing I. multifiliis.

1. Introduction

Ichthyophthirius multifiliis Fouquet, 1876, is one of the most harmful parasitic pathogens in freshwater teleosts, with low host specificity [1,2]. It causes ichthyophthiriasis, commonly known as the “white spot disease”, which can cause great economic losses to the global aquaculture industry [1,2,3]. It has four developmental stages (Figure 1): a parasitic trophont, a free-swimming protomont, a reproductive tomont, and an infective theront [3,4]. When the trophont matures, it naturally sheds itself from the host and becomes a free-swimming protomont; when the protomont attaches itself to a flat surface, it soon transforms into a tomont. The tomont then undergoes binary fission, producing hundreds of daughter cells called tomites, which further form infectious theronts. Theronts invade the epidermis of susceptible fish, and then rapidly transform into trophonts to complete their entire life cycle [2,4,5,6].
The prevention and control of ichthyophthiriasis have long been a key issue to fish parasite disease. Malachite green, as the most effective parasiticide to treat ichthyophthiriasis [7], has been banned in aquaculture practice because of its teratogenic, genotoxic, and carcinogenic effects [8,9,10]. Meanwhile, other chemicals, such as formalin [11], copper sulphate [12], potassium permanganate [13], and so on, have raised some environmental concerns [6]. Therefore, it is necessary to search for efficient, alternative, and environmentally friendly products to control I. multifiliis infection.
Herbal medicines have the characteristics of easy biodegradation and a low environmental hazard, which, in general, makes them environmentally friendly [14]. Multiple studies have shown that some plants possess effective parasiticide properties, and that some plant extracts show high efficacy against the free-swimming stages of I. multifiliis [5,15,16,17,18,19,20]. Berberine (BBR, C20H19NO5, MW 336.37) is an isoquinoline alkaloid and a positively charged particle isolated from several species of plants popular in Chinese herbal medicines, but most commonly from Coptis chinensis. Owing to its wide range of sources, low cost, and low toxicity, there have been many studies on the pharmacological effects of berberine, including the antibacterial [21], antineoplastic [22], and antiparasitic [23] properties. Given this background, we speculated that berberine may be an effective treatment against the free-living I. multifiliis (theronts and tomonts) in fish, but this hypothesis had not been tested before. In the present study, we used berberine and I. multifiliis to determine the efficacy and antiparasitic mechanism of berberine in vitro against the free-living I. multifiliis (theronts and protomonts).

2. Materials and Methods

2.1. Fish

Goldfish (Carassius auratus) weighing 14.2 ± 1.4 g (mean ± SD) were obtained from a local ornamental fish shop in Wuhan, China. All fish were distributed into several 30 L transparent tanks, and the tanks were kept under a flow system with dissolved oxygen at 6.0–8.0 mg/L, pH at 7.4 ± 0.2, a constant temperature of 23.0 ± 0.5 °C, and a light:dark period of 12:12 h. The fish were fed 1% body weight once a day with commercial fish pellet feedstuffs (Wuhan, China). These goldfish were partly used for propagation of I. multifiliis to obtain experimental protomonts and theronts, and partly used for the testing of 96 h acute toxicity of berberine to goldfish.

2.2. Parasites

Ichthyophthirius multifiliis was isolated from the goldfish. Several heavily infected fish were placed into 500 mL of aerated water for 30 min, which caused mature trophonts to fall off the fish. The unsettled protomonts were either directly randomly assigned into 24-well microtiter plates for medicinal tests or collected into 35 mm Petri dishes containing distilled water to hatch into theronts in a 23.0 ± 0.5 °C incubator.

2.3. Berberine

Berberine with a purity greater than 97% (HPLC) was purchased from Macklin (Shanghai, China). Before the experiments, berberine was mixed with distilled water to formulate into a series of stock solutions with different concentrations of 2, 5, 10, 15, 20, 30, and 40 mg/L, stored at 4 °C in a refrigerator, and settled in room temperature before use.

2.4. Effect of Berberine on I. multifiliis Theronts

In vitro studies that were conducted to determine the antiprotozoal activity of berberine against I. multifiliis theronts were adapted from the previously devised method [12,15,24]. Theront concentration was determined by taking 100 μL droplets from the theront suspension and calculating the average of five counts via a Sedgewick Rafter counting chamber and a microscope (40× magnification) [24,25]. Then, 100 μL of theront solution per well was placed into a 96-well microtiter plate and exposed to 100 μL berberine solutions, so that the final concentrations in the 96-well plate were 0, 1.0, 2.5, 5.0, 7.5, 10.0, 15.0, and 20.0 mg/L. Acute toxicity was assessed by the mortality of theronts 4 h after the treatment; theronts with an absence of motility and abnormal morphology were considered dead. The trial was conducted at 23.0 ± 0.5 °C and repeated three times.

2.5. Effect of Berberine on I. multifiliis Protomonts

According to the method described above, an in vitro study was designed to assess the efficacy of berberine against I. multifiliis tomonts. Protomonts that had discarded fish mucus were distributed into a 24-well microtiter plate, at the concentration of 30 protomonts (with 1000 μL of distilled water) per well. Beforehand, 1000 μL of prepared stock solutions was added to each well, so that the final concentrations of each well were 0, 1.0, 2.5, 5.0, 7.5, 10.0, 15.0, and 20.0 mg/L. Distilled water was used for the control group. After 4 h, the acute toxicity of berberine to protomonts was directly assessed by the number of dead protomonts. After 20 h of treatment, the percentage of theronts released from tomonts was assessed via microscopic examination. The mortality of protomonts was determined by their cytoplasmic immobility and abnormal morphology. The experiment was conducted at 23.0 ± 0.5 °C, and all groups were tested three times.

2.6. Ultrastructural Analysis

Berberine-treated for 4 h and untreated (control group) Ichthyophthirius multifiliis protomonts were fixed with 0.1 M phosphate-buffered 2.5% glutaraldehyde (pH 7.2) over 12 h at 4 °C. The parasite samples were processed according to the method described by Ozaki et al. [26]. Ultrastructural analysis was performed using a Hitachi 7700 transmission electron microscope (Tokyo, Japan) at 80.0 kV acceleration voltage.

2.7. Acute Toxicity of Berberine to Goldfish

The acute toxicity of berberine to goldfish was determined by the method of aqueous static renewal 96 h bioassays. Goldfish were divided into five aquariums (10 fish/aquarium), and a series of berberine solutions with different concentrations of 0 (control group), 300, 400, 500, and 600 mg/L were prepared for goldfish aqueous exposures. Mortality observations and recordings of goldfish in the aquariums were taken every 24 h. The fish were not fed during the exposures.

2.8. Statistical Analysis

Statistical analyses were performed using SPSS 18.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 8.0 (Graphpad Inc., San Diego, CA, USA). The data of in vitro trials were analyzed using a nonparametric method (the Kruskal–Wallis test), and significant differences between treatment and control groups were estimated using a one-way ANOVA (significant differences p < 0.05), followed by Dunnett’s test for multiple comparisons. The EC50 values and their 95% confidence intervals were calculated by the GraphPad Prism 8.0 dose procedure, and the LC50 values and their 95% confidence intervals were calculated by the SPSS 18.0 probit procedure.

3. Results

3.1. Acute Toxicity of Berberine to I. multifiliis Theronts and Protomonts

The mortality of theronts at a concentration of 0 mg/L was 18.43 ± 3.97% (Table 1). The mortality of theronts at a concentration of 5 mg/L was significantly higher (43.10 ± 3.35%), and the administration of 15 mg/L of berberine for 4 h killed all I. multifiliis theronts (100%).
The efficacy of berberine against protomonts was low: at 20 mg/L berberine concentration, their mortality was only 4.44 ± 1.92% (Table 1). However, berberine was effective at obstructing the generation of tomonts: at a low concentration of 2.5 mg/L, only 37.51 ± 3.81% theronts were released from tomonts, and at concentrations greater than 5 mg/L, no tomonts transformed into theronts (Table 1).

3.2. Ultrastructural Observation

The I. multifiliis protomonts in the control group were smooth and round (Figure 2A). The mitochondria were spherical to ovoid in shape (0.77–1.09 μm in length and 0.27–0.44 μm in width), and the tubular structures of their inner membrane were intact (Figure 2B). Ribosomes were scattered around the cytoplasm (Figure 2C).
After treatment with berberine, I. multifiliis protomonts exhibited several signs of damage. The cells were deformed and showed cytoplasmic vacuolation (Figure 2D), the inner membrane of the mitochondria was destroyed (Figure 2E), and the mitochondria exhibited considerable swelling after 4 h of treatment (0.62–0.89 μm in length and 0.44–0.63 μm in width). The number of ribosomes was decreased significantly in the cytoplasm (Figure 2F). Moreover, a great deal of organelle fragments were observable in the cytoplasm of berberine-treated protomonts (Figure 2F).

3.3. Acute Toxicity of Berberine to Goldfish

The goldfish exposed to various concentrations of berberine showed different responses to treatment within 96 h post-treatment (Table 2). Goldfish did not die under the treatment with berberine at concentrations of 0 (control) or 300 mg/L. The fish treated with berberine at 400 or 500 mg/L showed a mortality rate of 30%, and 70% of goldfish died when treated with 600 mg/L of berberine within 96 h (Table 2). The 96 h LC50 value for berberine in goldfish was 528.44 mg/L. (Table 3).

4. Discussion

The disease caused by I. multifiliis probably brings about more damage to freshwater fish than any other eukaryotic pathogen [2,3]. There are two main strategies for the treatment of the disease: one is aiming at the parasitic trophont stage within the host′s epithelium, while the other is targeting the external stages where the parasite is free-living [2,4]. However, the trophonts in the epithelium are protected both by the mucus secreted by the fish and the proliferation of epithelial tissue, so it is difficult for drugs to kill trophonts through the epithelial tissue.
Straus and Griffin [24] argued that killing infectious theronts could prevent the disease from spreading to other fish. Several studies reported that theronts are more sensitive than other stages of I. multifiliis to plant extracts [5,16,20] and chemicals [11,15]. Killing theronts can effectively prevent fish from being infected by I. multifiliis. It is therefore of high priority to access the antiprotozoal activity of berberine against I. multifiliis theronts. Our results showed that berberine could kill almost all theronts at a concentration of 15 mg/L in 4 h, and berberine concentrations of 7.5 mg/L and more had over 60% efficiency in killing I. multifiliis theronts. In comparison, it took 4 h to kill all theronts using commercially available copper naphthenate at a concentration of 8 mg/L (organic copper content = 2.4%) [27]. The concentrations of ethanol extracts of Zingiber officinale and Cynanchum atratum needed to achieve 100% theronts mortality were 8 mg/L and 16.0 mg/L, respectively [28]. The concentration of berberine needed to achieve 100% theront mortality at the 4 h time point was much lower than that of garlic extract, which required 62.5 mg/L for 15 h [15]. However, 0.6 mg/L of magnolol managed to eliminate all theronts after 4 h [29]. Therefore, berberine could effectively eliminate theronts in water.
Although tomonts do not directly invade fish, they can produce a large number of infective theronts [2]. Therefore, it is important to stop theront infection by preventing the productive tomonts. At a concentration of 20 mg/L berberine could kill all theronts quickly, but only killed 4.44% of protomonts, which indicated that protomonts are more resistant to berberine than theronts. This is similar to results obtained using other plant extracts [20,30]. Even so, at a concentration of 5 mg/L for 4 h, berberine could completely prevent tomonts from releasing theronts. This suggests that berberine may effectively block the life cycle of I. multifiliis even at low concentrations, and thus significantly decrease the re-infection with theronts and increase the survival rate of infected fish.
Previous studies have shown that Chinese herbal extracts not only have a good in vitro anti-I. multifiliis effect, but also have low toxicity to fish, whose LC50 is generally several to dozens of times higher than that of the EC50 against theronts. For example, psoralen at a concentration of 0.8 mg/L can kill all I. multifiliis theronts within 4 h, but the LC50 at 96 h is 5.6 mg/L [31]. For magnolol, the 96 h LC50 value for goldfish is 6.02 mg/L, but only 0.6 mg/L of magnolol can kill all theronts in 4 h [29]. In our study, the LC50 (528.44 mg/L) for goldfish was about 67 times larger than the EC50 (7.86 mg/L) for killing theronts, and 192 times higher than the EC50 (2.75 mg/L) for inhibiting the development of cysts into theronts (Table 3). The results showed that berberine is an effective and safe drug against I. multifiliis in vitro.
Furthermore, our TEM results demonstrated that berberine treatment resulted in the damage of organelles and cytomembranes, reflected in the reduction in the number of ribosomes, the rupturing of the inner membrane of mitochondria, and mitochondrial swelling. This is similar to the results of Qu′s and Ruider′s studies [7,32]. Ribosomes are critical in protein production in all cells, and the I. multifiliis is no exception. Their major function is to promote the translational process, and mutations or alterations of ribosomal proteins cause abnormal cell proliferation [33,34]. Mohammad et al. [35] reported that one of the targets of aminoglycosides was the ribosomes of Leishmania. Therefore, we suspected that the antiparasitic function of berberine may relate to the decrease in the number of ribosomes in I. multifiliis. Mitochondrial changes may also be directly involved in the cell death process of I. multifiliis. Some studies indicated that berberine could inhibit various human tumor cells by inducing mitochondrial apoptosis [36,37]. In addition, several reports provided evidence that mitochondria are one of the primary cellular targets of berberine in human tumor cells [36,37,38]. Our results suggested that berberine causes structural alterations in parasite cells, particularly severe disruption to the inner membrane of mitochondria. These effects are similar to those achieved by the treatment with malachite green [7,32] and 10-gingerol [39]. In Fu’s study, 10-gingerol reduced ATP ase activities, leading to the accumulation of free radicals, which further led to the destruction of cell membranes and organelles. [39] The mechanism by which berberine kills I. multifiliis may be similar to that of 10-gingerol, but more studies are needed to test this hypothesis.

Author Contributions

M.L. designed the experiments. K.H. performed the experiments, analyzed the data, and wrote the paper. G.H. and R.W. helped with the data analysis. Q.Z., W.L., S.W., H.Z. and G.W. reviewed and revised the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the earmarked fund for CARS, grant number CARS-45; the open project of Hubei Key Laboratory of Three Gorges Project for Conservation of Fishes, grant number 2021050-ZHX; the National Natural Science Foundation of China, grant number 32170437; the Second Tibetan Plateau Scientific Expedition and Research Program (STEP), grant number 2019QZKK0304; the Protist 10,000 Genomics Project (P10K) Consortium; and the National Aquatic Biological Resource Center (NABRC).

Institutional Review Board Statement

The animal study protocol was approved by the Animal Ethics Committee of the Institute of Hydrobiology, Chinese Academy of Sciences (project identification code: IHB/LL/2020025; date of approval: 27 July 2022).

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We especially thank Z.X. and Y.X., Analysis and Testing Center, Institute of Hydrobiology, CAS, for their technical support in TEM observation. We are also thankful to Ivan Jakovlić for polishing the language.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Buchmann, K.; Sigh, J.; Nielsen, C.; Dalgaard, M. Host responses against the fish parasitizing ciliate Ichthyophthirius multifiliis. Vet. Parasitol. 2001, 100, 105–116. [Google Scholar] [CrossRef]
  2. Matthews, R.A. Ichthyophthirius multifiliis Fouquet and ichthyophthiriosis in freshwater teleosts. In Advances in Parasitology; Baker, J.R., Muller, R., Rollinson, D., Eds.; Elsevier: Amsterdam, The Netherlands, 2005; Volume 59, pp. 159–241. [Google Scholar]
  3. Hines, R.S.; Spira, D.T. Ichthyophthirius multifiliis (Fouquet) in the mirror carp, Cyprinus carpio L. I. Course of infection. J. Fish Biol. 1973, 5, 385–392. [Google Scholar]
  4. Li, M.; Bastos Gomes, G.; Zhao, W.; Hu, G.; Huang, K.; Yoshinaga, T.; Clark, T.G.; Li, W.; Zou, H.; Wu, S.; et al. Cultivation of fish ciliate parasites: Progress and prospects. Rev. Aquac. 2022. [Google Scholar] [CrossRef]
  5. Ling, F.; Lu, C.; Tu, X.; Yi, Y.; Huang, A.; Zhang, Q.; Wang, G. Antiprotozoal screening of traditional medicinal plants: Evaluation of crude extract of Psoralea corylifolia against Ichthyophthirius multifiliis in goldfish. Parasitol. Res. 2013, 112, 2331–2340. [Google Scholar] [CrossRef]
  6. Picon-Camacho, S.M.; Marcos-Lopez, M.; Bron, J.E.; Shinn, A.P. An assessment of the use of drug and non-drug interventions in the treatment of Ichthyophthirius multifiliis Fouquet, 1876, a protozoan parasite of freshwater fish. Parasitology 2012, 139, 149–190. [Google Scholar] [CrossRef]
  7. Ruider, S.; Schmahl, G.; Mehlhorn, H.; Schmidt, H.; Ritter, G. Effects of different malachite green derivatives and metabolites on the fish ectoparasite, Ichthyophthirius multifiliis, Fouquet 1876 (Hymenostomatida, Ciliophora). Eur. J. Protistol. 1997, 33, 375–388. [Google Scholar] [CrossRef]
  8. Alderman, D.J. Malachite green a review. J. Fish Dis. 1985, 8, 289–298. [Google Scholar] [CrossRef]
  9. Srivastava, S.; Sinha, R.; Roy, D. Toxicological effects of malachite green. Aquat. Toxicol. 2004, 66, 319–329. [Google Scholar] [CrossRef]
  10. Wahli, T.; Schmitt, T.; Meier, W. Evaluation of alternatives to malachite green oxalate as a therapeutant for ichthyophthiriosis in rainbow-trout, Oncorhynchus mykiss. J. Appl. Ichthyol. 1993, 9, 237–249. [Google Scholar] [CrossRef]
  11. Rowland, S.J.; Mifsud, C.; Nixon, M.; Read, P.; Landos, M. Use of formalin and copper to control ichthyophthiriosis in the Australian freshwater fish silver perch (Bidyanus bidyanusMitchell). Aquac. Res. 2008, 40, 44–54. [Google Scholar] [CrossRef]
  12. Ling, K.H.; Sin, Y.M.; Lam, T.J. Effect of copper sulphate on ichthyophthiriasis (white spot disease) in goldfish (Carassius auratus). Aquaculture 1993, 118, 23–35. [Google Scholar] [CrossRef]
  13. Straus, D.L.; Griffin, B.R. Efficacy of Potassium Permanganate in Treating Ichthyophthiriasis in Channel Catfish. J. Aquat. Anim. Health 2002, 14, 145–148. [Google Scholar] [CrossRef]
  14. Valladao, G.M.; Gallani, S.U.; Pilarski, F. Phytotherapy as an alternative for treating fish disease. J. Vet. Pharm. 2015, 38, 417–428. [Google Scholar] [CrossRef] [PubMed]
  15. Buchmann, K.; Jensen, P.B.; Kruse, K.D. Effects of Sodium Percarbonate and Garlic Extract on Ichthyophthirius multifiliis Theronts and Tomocysts: In Vitro Experiments. N. Am. J. Aquac. 2003, 65, 21–24. [Google Scholar] [CrossRef]
  16. Chao, C.; Qizhong, Z.; Fen, L. Effect of twenty Chinese herbal medicines on killing trophonts, cysts and theronts of Ichthyophthirius multifiliis in vitro. Freshw. Fish. 2010, 40, 55–60. [Google Scholar]
  17. Ekanem, A.P.; Obiekezie, A.; Kloas, W.; Knopf, K. Effects of crude extracts of Mucuna pruriens (Fabaceae) and Carica papaya (Caricaceae) against the protozoan fish parasite Ichthyophthirius multifiliis. Parasitol. Res. 2004, 92, 361–366. [Google Scholar] [CrossRef]
  18. Yao, J.Y.; Shen, J.Y.; Li, X.L.; Xu, Y.; Hao, G.J.; Pan, X.Y.; Wang, G.X.; Yin, W.L. Effect of sanguinarine from the leaves of Macleaya cordata against Ichthyophthirius multifiliis in grass carp (Ctenopharyngodon idella). Parasitol. Res. 2010, 107, 1035–1042. [Google Scholar] [CrossRef]
  19. Yao, J.Y.; Zhou, Z.M.; Li, X.L.; Yin, W.L.; Ru, H.S.; Pan, X.Y.; Hao, G.J.; Xu, Y.; Shen, J.Y. Antiparasitic efficacy of dihydrosanguinarine and dihydrochelerythrine from Macleaya microcarpa against Ichthyophthirius multifiliis in richadsin (Squaliobarbus curriculus). Vet. Parasitol. 2011, 183, 8–13. [Google Scholar] [CrossRef]
  20. Yi, Y.L.; Lu, C.; Hu, X.G.; Ling, F.; Wang, G.X. Antiprotozoal activity of medicinal plants against Ichthyophthirius multifiliis in goldfish (Carassius auratus). Parasitol. Res. 2012, 111, 1771–1778. [Google Scholar] [CrossRef]
  21. Li, T.; Wang, P.; Guo, W.; Huang, X.; Tian, X.; Wu, G.; Xu, B.; Li, F.; Yan, C.; Liang, X.; et al. Natural Berberine-Based Chinese Herb Medicine Assembled Nanostructures with Modified Antibacterial Application. ACS Nano 2019, 13, 6770–6781. [Google Scholar] [CrossRef]
  22. Xing, Y.; Liu, X.; Lin, Y.; Zhang, Y. Progress in pharmacological effects and clinical applications of berberine. Chin. J. Pharmacol. Toxicol. 2017, 31, 491–502. [Google Scholar] [CrossRef]
  23. Chang, Y.; Mei, X.; Hou, Y.; Song, J.; Lv, F. Research progress of berberine and its derivatives against parasite infection. Chin. J. Endem. 2020, 39, 457–462. [Google Scholar]
  24. Straus, D.L.; Griffin, B.R. Prevention of an Initial Infestation of Ichthyophthirius multifiliis in Channel Catfish and Blue Tilapia by Potassium Permanganate Treatment. N. Am. J. Aquac. 2001, 63, 11–16. [Google Scholar] [CrossRef]
  25. Schlenk, D.; Gollon, J.L.; Griffin, B.R. Efficacy of Copper Sulfate for the Treatment of Ichthyophthiriasis in Channel Catfish. J. Aquat. Anim. Health 1998, 10, 390–396. [Google Scholar] [CrossRef]
  26. Ozaki, K.; Ito, E.; Tanabe, S.; Natsume, K.; Tsuji, K.; Harada, K. Electron Microscopic Study on Lysis of a Cyanobacterium Microcystis. J. Health Sci. 2009, 55, 578–585. [Google Scholar] [CrossRef] [Green Version]
  27. Hu, G.R.; Zhao, W.S.; Wang, R.Q.; Zou, H.; Li, W.X.; Wu, S.G.; Li, M.; Wang, G.T. In vitro assessment of copper naphthenate against the free-living stages of Ichthyophthirius multifiliis. Aquac. Rep. 2020, 17, 100404. [Google Scholar] [CrossRef]
  28. Lin, D.J.; Hua, Y.N.; Zhang, Q.Z.; Xu, D.H.; Fu, Y.W.; Liu, Y.M.; Zhou, S.Y. Evaluation of medicated feeds with antiparasitical and immune-enhanced Chinese herbal medicines against Ichthyophthirius multifiliis in grass carp (Ctenopharyngodon idellus). Parasitol. Res. 2016, 115, 2473–2483. [Google Scholar] [CrossRef]
  29. Song, C.; Song, K.; Wu, X.; Tu, X.; Qi, X.; Wang, G.; Ling, F. Antiparasitic efficacy and safety assessment of magnolol against Ichthyophthirius multifiliis in goldfish. Aquaculture 2018, 486, 9–17. [Google Scholar] [CrossRef]
  30. Zhang, Q.; Xu, D.H.; Klesius, P.H. Evaluation of an antiparasitic compound extracted from Galla chinensis against fish parasite Ichthyophthirius multifiliis. Vet. Parasitol. 2013, 198, 45–53. [Google Scholar] [CrossRef]
  31. Song, K.; Ling, F.; Huang, A.; Dong, W.; Liu, G.; Jiang, C.; Zhang, Q.; Wang, G. In vitro and in vivo assessment of the effect of antiprotozoal compounds isolated from Psoralea corylifolia against Ichthyophthirius multifiliis in fish. Int. J. Parasitol. Drugs Drug Resist. 2015, 5, 58–64. [Google Scholar] [CrossRef] [Green Version]
  32. Qu, S.; Song, C.; Tan, X.; Wang, G.; Ling, F. Comparative proteomic analysis provides insight into the key proteins as potential targets underlying the effect of malachite green against Ichthyophthirius multifiliis. J. Fish Dis. 2021, 44, 881–892. [Google Scholar] [CrossRef] [PubMed]
  33. Guo, X.; Shi, Y.; Gou, Y.; Li, J.; Han, S.; Zhang, Y.; Huo, J.; Ning, X.; Sun, L.; Chen, Y.; et al. Human ribosomal protein S13 promotes gastric cancer growth through down-regulating p27(Kip1). J. Cell Mol. Med. 2011, 15, 296–306. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Pelchovich, G.; Nadejda, S.; Dana, A.; Tuller, T.; Bravo, I.G.; Gophna, U. Ribosomal mutations affecting the translation of genes that use non-optimal codons. FEBS J. 2014, 281, 3701–3718. [Google Scholar] [CrossRef] [PubMed]
  35. Mohammad, M.; Francoise, L.; Simon, L.C.; Malka, R.G. Ribosomes of Leishmania are a target for the aminoglycosides. Parasitol. Res. 1995, 81, 421–425. [Google Scholar] [CrossRef]
  36. Li, L.; Wang, X.; Sharvan, R.; Gao, J.; Qu, S. Berberine could inhibit thyroid carcinoma cells by inducing mitochondrial apoptosis, G0/G1 cell cycle arrest and suppressing migration via PI3K-AKT and MAPK signaling pathways. Biomed. Pharmacother. 2017, 95, 1225–1231. [Google Scholar] [CrossRef]
  37. Yao, Z.; Wan, Y.; Li, B.; Zhai, C.; Yao, F.; Kang, Y.; Liu, Q.; Lin, D. Berberine induces mitochondrialmediated apoptosis and protective autophagy in human malignant pleural mesothelioma NCIH2452 cells. Oncol. Rep. 2018, 40, 3603–3610. [Google Scholar] [CrossRef] [Green Version]
  38. Kuo, H.P.; Lee, Y.J.; Hsu, C.Y.; Lee, S.L.; Hsu, S.C.; Chuang, T.C.; Liu, J.Y.; Kuo, C.L.; Ho, C.T.; Kao, M.C. Growth-suppressive effect of berberine on endometrial carcinoma cells: Role of mitochondrial and PI3K/Akt pathway. J. Funct. Foods 2015, 17, 600–609. [Google Scholar] [CrossRef]
  39. Fu, Y.W.; Wang, B.; Zhang, Q.Z.; Xu, D.H.; Liu, Y.M.; Hou, T.L.; Guo, S.Q. Efficacy and antiparasitic mechanism of 10-gingerol isolated from ginger Zingiber officinale against Ichthyophthirius multifiliis in grass carp. Vet. Parasitol. 2019, 265, 74–84. [Google Scholar] [CrossRef]
Pathogens 11 01207 g001 550
Figure 1. The life cycle of Ichthyophthirius multifiliis: (1) The infectious theront invades the epidermis of fish and grows into a trophont. (2) Mature trophont leaves the host to become a free-swimming protomont. (3) Protomont secretes a cyst wall to become a tomont. (4) Tomont undergoes binary fission, producing large numbers of theronts to invade fish again (cited from Li et al. [4]).
Figure 1. The life cycle of Ichthyophthirius multifiliis: (1) The infectious theront invades the epidermis of fish and grows into a trophont. (2) Mature trophont leaves the host to become a free-swimming protomont. (3) Protomont secretes a cyst wall to become a tomont. (4) Tomont undergoes binary fission, producing large numbers of theronts to invade fish again (cited from Li et al. [4]).
Pathogens 11 01207 g001
Pathogens 11 01207 g002 550
Figure 2. Transmission electron micrographs of untreated (A) and 4 h treated (D) I. multifiliis trophonts. (A) The overall structure of untreated trophont. Scale bar = 10 µm (B) Normal mitochondria and large numbers of ribosomes in the control group. Scale bar = 500 nm. (C) Large numbers of ribosomes in the control group. Scale bar = 500 nm. (D) The overall structure of a trophont treated for 4 h with berberine. Scale bar = 10 µm. (E) Destruction of mitochondrial inner membrane and reduction in the number of ribosomes in the berberine-treated group. Scale bar = 500 nm. (F) Reduction in the number of ribosomes and organelle fragments in the berberine-treated group. Scale bar = 500 nm. Abbreviations: CM, cell membrane; V, vacuole; Mit, mitochondrion; IM, inner membrane; OM, outer membrane; R, ribosome; OF, organelle fragment.
Figure 2. Transmission electron micrographs of untreated (A) and 4 h treated (D) I. multifiliis trophonts. (A) The overall structure of untreated trophont. Scale bar = 10 µm (B) Normal mitochondria and large numbers of ribosomes in the control group. Scale bar = 500 nm. (C) Large numbers of ribosomes in the control group. Scale bar = 500 nm. (D) The overall structure of a trophont treated for 4 h with berberine. Scale bar = 10 µm. (E) Destruction of mitochondrial inner membrane and reduction in the number of ribosomes in the berberine-treated group. Scale bar = 500 nm. (F) Reduction in the number of ribosomes and organelle fragments in the berberine-treated group. Scale bar = 500 nm. Abbreviations: CM, cell membrane; V, vacuole; Mit, mitochondrion; IM, inner membrane; OM, outer membrane; R, ribosome; OF, organelle fragment.
Pathogens 11 01207 g002
Table
Table 1. Efficacy of berberine against I. multifiliis theronts and tomonts.
Table 1. Efficacy of berberine against I. multifiliis theronts and tomonts.
Concentration (mg/L)TherontsTomonts
MT (%)MT (%)PRT (%)
018.43 ± 3.97 a0.00 ± 0.00 a100.00 ± 0.00 a
123.11 ± 17.02 a0.00 ± 0.00 a93.33 ± 3.33 b
2.530.82 ± 18.36 ab0.00 ± 0.00 a37.51 ± 3.81 c
543.10 ± 3.35 b0.00 ± 0.00 a0.00 ± 0.00 d
7.565.41 ± 3.48 c0.00 ± 0.00 a0.00 ± 0.00 d
1072.66 ± 7.05 c0.00 ± 0.00 a0.00 ± 0.00 d
1599.30 ± 1.21 d0.00 ± 0.00 a0.00 ± 0.00 d
20100.00 ± 0.00 d4.44 ± 1.92 b0.00 ± 0.00 d
MT: mortality of theronts and tomonts after 4 h. PRT: percentage of theronts released from tomonts after 20 h. The mortality data are given as a mean ± standard deviation. In the same column, the values with different lowercase letters (a, b, c, d) are significantly different (p < 0.05).
Table
Table 2. Acute toxicity of berberine to goldfish in aqueous static renewal 96 h bioassays.
Table 2. Acute toxicity of berberine to goldfish in aqueous static renewal 96 h bioassays.
Concentration (mg/L)Total No. TestedNo. of DeadSurvival (%)
24 h48 h72 h96 h
Control100000100
300100000100
40010003070
50010030070
60010402130
Table
Table 3. In vitro anti-theront efficacy and acute toxicity of berberine to healthy goldfish.
Table 3. In vitro anti-theront efficacy and acute toxicity of berberine to healthy goldfish.
In Vitro Anti-Theront Efficacy at 4 hAcute Toxicity to Goldfish at 96 h
EC5095% Confidence
Interval		LC5095% Confidence
Interval
7.866.61–14.92528.44459.07–713.70
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Huang, K.; Hu, G.; Wang, R.; Zeng, Q.; Li, W.; Zou, H.; Wu, S.; Wang, G.; Li, M. In Vitro Assessment of Berberine against Ichthyophthirius multifiliis in Goldfish. Pathogens 2022, 11, 1207. https://doi.org/10.3390/pathogens11101207

AMA Style

Huang K, Hu G, Wang R, Zeng Q, Li W, Zou H, Wu S, Wang G, Li M. In Vitro Assessment of Berberine against Ichthyophthirius multifiliis in Goldfish. Pathogens. 2022; 11(10):1207. https://doi.org/10.3390/pathogens11101207

Chicago/Turabian Style

Huang, Ke, Guangran Hu, Runqiu Wang, Qingwen Zeng, Wenxiang Li, Hong Zou, Shangong Wu, Guitang Wang, and Ming Li. 2022. "In Vitro Assessment of Berberine against Ichthyophthirius multifiliis in Goldfish" Pathogens 11, no. 10: 1207. https://doi.org/10.3390/pathogens11101207

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop