Anthelmintic effects of peppermint (Mentha piperita), lemon (Citrus limon), and tea tree (Melaleuca alternifolia) essential oils against Monogenean parasite (Dactylogyrus sp.) on carp (Cyprinus carpio)
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Introduction
The aquaculture industry has grown significantly in the world from 17.3 million tons of average annual production in the 1990s to 122.6 million tons in 2020. Carp is an economically important species in the industry and in 2020 common carp was ranked fourth worldwide in total finfish production in inland aquaculture (FAO, 2022). The rapid increase in production to meet multiplying demand has elevated disease risks by increasing stress factors such as suboptimal water quality, inadequate nutrition, and higher stocking density in aquaculture conditions. Ectoparasitic infections, particularly by monogeneans, have resulted in significantly greater losses in the fish culture industry (Ernst et al., 2002; Shinn et al., 2015).
Monogeneans are a group of parasitic flatworms that are commonly found on the skin or the gills of fish. These parasites are known for their host specificity, which means they tend to infect only one or a few closely related species of fish. Host specificity helps to prevent competition with other monogenean species that infect different fish hosts. Their short and direct life cycle induces the rapid invasion in fish gills or skin (Ernst et al., 2002). One of the Monogenean ectoparasites, the Dactylogyrus species exhibits hermaphroditism and oviparity. Their free-swimming ciliated larvae (oncomiracidia) emerge directly from eggs and are capable of infecting fish without the need for an intermediary host (Zoral et al., 2017). Dactylogyrus sp. causes serious damage in fish by triggering pathological changes that inhibit gas exchange in gills resulting in appetite loss, low growth performance, and high mortality rates (Obiekezie & Taege, 1991). In tissues damaged by parasites, secondary infections due to bacterial and fungal pathogens are also common (Reed et al., 2009).
A common treatment technique has been the application of chemicals like formalin, copper sulfate, potassium permanganate, and hydrogen peroxide on fish by the bath. While these chemicals can be effective, their use can negatively impact water quality, and the toxins they leave behind in fish pose risks to human health (Ling et al., 2015; Hashimoto et al., 2016; Soares et al., 2016; Soares et al., 2017a, b). For these reasons, the use of chemicals against fish diseases has been restricted in many countries (Ling et al., 2015). For example, inside the European Union, some of the chemical therapeutics such as malachite green, or methylene blue are already banned (Lieke et al., 2020). The research of alternative treatments for parasitic infections in fish has been prompted by these problems (Lieke et al., 2020; Yildiz & Bekcan, 2020; Phan et al., 2021).
Plant essential oils have been gaining interest in aquaculture as a natural alternative to antibiotics and other synthetic drugs for controlling diseases and improving growth performance in farmed aquatic animals. Essential oils are volatile, aromatic compounds extracted from different parts of plants, such as leaves, flowers, and fruits, that possess diverse biological activities, including antimicrobial, antiviral, antiparasitic, antioxidant, and immunostimulatory properties. Over the past few years, there has been an increasing interest in searching alternative treatment options for various fish parasites, as well as studying the cytotoxic properties of different herbal products such as plant extracts, essential oils, and bioactive metabolites derived from diverse plant sources. Some of these studies have yielded promising results with regard to their antiparasitic effects (Wang et al., 2011; Wu et al., 2011; Lu et al., 2012; Ramudu & Dash, 2013; Valladao et al., 2015; Tavares-Dias, 2018). Thus, herbal treatment emerged as having potential as an innovative method against various parasites in aquaculture (Soares & Tavares-Dias, 2013; Soares et al., 2016; Soares et al., 2017a, b).
Of the plants tested for their antiparasitic potential in this study, peppermint is used in medicine as an antimicrobial and an antioxidant. The main component of its essential oil is menthol (Kumar et al., 2012; Freire et al., 2012; Tsai et al., 2013). Lemon essential oil's antibacterial, antioxidant, and anticancer properties are due to the phenolic compounds it contains, especially limonin (Bulfon et al., 2015). Tea tree is an Australian plant containing the main metabolite of terpinen-4-ol and has a wide range of antiseptic, antimicrobial, anti-inflammatory, and antiparasitic activity (Thomsen et al., 2011). Peppermint and lemon essential oil have not been tested for their anti-helminthic activities in fish before to our knowledge. Tea tree oil has been tested on Gyrodactylus spp. infection of the three-spined stickleback Gasterosteus aculeatus (Steverding et al., 2005).
Phytotherapeutic antiparasitic properties of various essential oils have been evaluated against diverse species of monogeneans. The effectiveness of these treatments varied between 0 to 100 %, depending on the specific essential oil and therapeutic approach utilized. Ling et al (2015) reported that cinnamic acid and cinnamaldehyde were 100 % effective against D. intermedius in Carassius auratus. On the other hand, Soares et al (2017a) showed that the essential oils (EOs) extracted from Lippia sidoides were ineffective against Anacanthorus spathulatus in Colossoma macropomum. Numerous studies have indicated that essential oils may have the potential to treat Dactylogyrus species (Monogeneans) in aquaculture (Ling et al., 2015; Luo et al., 2016; Wang et al., 2011; Wu et al., 2011; Yao et al., 2011; Phan et al., 2021). Nevertheless, it is surprising to note that only a limited number of essential oils have been extensively studied and subjected to controlled experimentation to demonstrate their anthelmintic activity, either through in vitro assays or through therapeutic baths (in vivo assays) for parasitized hosts in aquaculture (Tavares-Dias, 2018). Thus, to the best of our knowledge, the antiparasitic activity of peppermint, lemon, and tea tree essential oils against fish monogenean, Dactylogyrus sp. has not been reported so far. Therefore, the present study attempted to investigate the anthelmintic activity of essential oils of peppermint, lemon, and tea tree against the monogenean parasite (Dactylogyrus sp.) on carp.
Materials And Methods
Fish and parasites
Carp weighing 50 – 100 g in the aquaponic system in Ankara University, Department of Fisheries and Aquaculture were used as fish material in the research. The study was carried out between March 2020 and September 2020 at Ankara University, Department of Fisheries and Aquaculture, Fish Health Laboratory. During the research, a total of 40 fish were used. Fish were placed in 2 fiber tanks (80×60×50 cm) (20 fish in each tank) ventilated by a dry air motor. The water temperature was maintained at 20 – 22 °C, dissolved oxygen at 5.50 – 5.97 mg/L, and pH at 6.97 – 7. Fish were fed with commercial feed containing 45 % raw protein at a 2 % total body weight ratio.
For in vitro analysis, adult parasites were collected from the gills of heavily infected carp. Carp were anesthetized in 25 mg/l concentration of clove oil solution and ectoparasite specimens were collected by light scraping from the gill mucus of carp as previously described by Yildiz and Bekcan (2020). Samples taken from the gill mucus were taken on a slide and examined under a binocular microscope. During the examination of the samples for parasites, principles reported by Lom and Dykova (1992) were applied. Dactylogyrus sp. was identified by observing four eye spots and a pair of hooks (Malmberg, 1970; Bruno et al., 2006).
Essential Oils
In the study, the antiparasitic effect of peppermint (main bioactive component: menthol), lemon (main bioactive component: limonene), and tea tree essential oils (main bioactive component: terpinen-4-ol) were investigated. Essential oils were obtained from the spice sellers (trade name: Unyazici) in Ankara. Since essential oils are not soluble in water dimethyl sulfoxide (DMSO) was used as a solvent.
In vitro determination of the antiparasitic effect of essential oils on Dactylogyrus sp.
In the in vitro experiments, during which the lethal effect of essential oils on Dactylogyrus sp. was examined, parasites collected from the gills were counted and then subjected to peppermint, lemon, and tea tree essential oils with 10, 5, 2.5, and 1 μl/ml concentrations between 1 and 15 min. In the experiments, each essential oil concentration was tested on 10 parasites. Parasites were examined continuously for their motility and shrinkage under the microscope and the times to nonmotility were recorded. Parasites that did not move and respond when touched with a needle were reported as dead. In the control group, parasites were exposed to DMSO, the solvent of essential oils. Since DMSO itself can have a lethal effect on the parasite, various concentrations of DMSO have been tested before starting the experiments to discard the possible effects of DMSO. The concentration of DMSO and water at a ratio of 1:50 has been determined to have no effect on the parasites (Table 1).
In vitro cumulative mortality of Dactylogyrus sp. exposed to DMSO solution.
Concentration
Cumulative Mortality (%)
1 min
2 min
3 min
4 min
5 min
6 min
7 min
8 min
9 min
10 min
15 min
20 min
1/10
60
100
100
100
100
100
100
100
100
100
100
100
1/20
0
70
90
100
100
100
100
100
100
100
100
100
1/30
0
0
50
70
100
100
100
100
100
100
100
100
1/40
0
0
0
0
0
0
0
0
0
20
30
50
1/50
0
0
0
0
0
0
0
0
0
0
0
0
In vivo determination of the antiparasitic effect of essential oils on Dactylogyrus sp.
Before performing in vivo tests with carp, EC50 concentrations of each herbal essential oil were determined by in vitro tests. A total of 40 carp were used in in vivo tests. Each essential oil was applied as a single bath to the carp (N=10 for each essential oil application). The control group contained the same number of carp (N=10). EC50 concentrations of peppermint (2.53 μl/ml in 5 min exposure), lemon (0.73 μl/ml in 5 min exposure), and tea tree essential oils (0.30 μl/ml in 2 min exposure) were tested for the respective durations. Treatments were carried out in a 20 L plastic pot containing 5 L of herbal essential oil solutions.
Samples (0.01 g) collected from the gill mucus of each carp before and after bath treatment were placed on glass slides, and non-motile and motile parasites were counted under a light microscope (Nikon 120 model) at 25 Å ~ 10 magnifications.
The antiparasitic efficacy was estimated according to the methods described in the previous study (Zhou et al., 2021) and the following formula was used:
{\rm{Antiparasitic}}\;{\rm{Efficacy}}\; = \;(({\text{A - B}})/{\rm{ A}}) \times 100
A: Average number of live parasites in gill mucus before essential oil exposure
B: Average number of live parasites in gill mucus after essential oil exposure
Statistics
Probit analysis was applied to determine EC50 values in vitro tests (Finney, 1971) using NCSS 2020 statistics program. In in vitro tests, in order to determine the relationship between the mean time to death of parasites and the concentration of the essential oil applied, Pearson's correlation test was used. Statistical analysis for in vivo test results was carried out using one-way ANOVA. In all statistical tests, differences in the efficacies of essential oils were considered significant when p<0.05.
Ethical Approval and Informed Consent
Fish management and experimental protocols (with the reference number 2020-8-67) approved by the Ankara University Ethics Committee were complied with during the research.
Results
In vitro antiparasitic effect of essential oils on Dactylogyrus sp.
In the in vitro tests, the antiparasitic effects of peppermint, lemon, and tea tree essential oils were determined to be time and dose-dependent (p<0.05). Using Pearson's correlation test, the relationship between increasing essential oil concentrations and decreasing mean time to nonmotility of parasites was found to be significant (p<0.05).
In in vitro peppermint essential oil tests, cumulative mortality of Dactylogyrus sp. was observed to reach 100 % at 10 μl/ml in 2 min, at 5 μl/ml in 7 min, at 2.5 μl/ml in 8 min and at 1 μl/ml in 15 min (Fig. 1). The antiparasitic effect was observed to increase with increasing concentration and exposure duration.
Fig. 1.
In vitro cumulative mortality of Dactylogyrus sp. exposed to peppermint essential oil. Control: 1:50 DMSO.
In in vitro lemon essential oil tests, cumulative mortality was 100 % at 10 μl/ml in 3 min, at 5 μl/ml in 4 min, at 2.5 μl/ml in 6 min, and at 1 μl/ml in 10 min. At the lowest concentration (1 μl/ml) using the lemon essential oil, cumulative mortality of Dactylogyrus sp. reached 100 % in vitro after 10 min (Fig. 2). At the highest concentration (10 μl/ml) all parasites died in 3 min. The antiparasitic activity of lemon essential oil as well increased with concentration and exposure duration.
Fig. 2.
In vitro cumulative mortality of Dactylogyrus sp. exposed to lemon essential oil. Control: 1:50 DMSO
The antiparasitic effect of tea tree essential oil at 10, 5, 2.5, 1, and 0.5 μl/ml concentrations against Dactylogyrus sp. were assessed for exposure durations between 1 and 3 min in in vitro tests. In these tests, cumulative mortality was observed to reach 100 % at 10, 5, and 2.5 μl/ml concentrations in 1 min, at 1 μl/ml in 2 min, and at 0.5 μl/ml in 3 min. In the experiments with tea tree essential oil (10, 5, 2.5, 1, and 0.5 μl/ml), cumulative mortality of Dactylogyrus sp. reached 100 % in 3 min (Fig. 3). Like peppermint and lemon essential oils, tea tree essential oil demonstrated an increasing antiparasitic effect with increasing concentration and exposure duration.
Fig. 3.
In vitro cumulative mortality of Dactylogyrus sp. exposed to tea tree essential oil. Control: 1:50 DMSO
Dactylogyrus sp. behavior after being exposed in vitro to peppermint, lemon, and tea tree essential oils
In in vitro experiments, Dactylogyrus sp. was observed to contract rapidly and die after exposure to peppermint, lemon, and tea tree essential oils (Fig. 4).
Fig. 4.
Parasite behavior; (a) Dactylogyrus sp. before exposure (alive); (b) Contraction of Dactylogyrus sp. after exposure (c) Death of Dactylogyrus sp. after exposure.
In vivo antiparasitic effect of essential oils on Dactylogyrus sp.
In in vivo tests, EC50 for peppermint (2.53 μl/ml) and lemon (0.73 μl/ml) essential oils in 5 min exposure and EC50 for tea tree (0.30 μl/ml) essential oil in 2 min exposure were applied as a single bath on carp and resulted in a significant reduction in parasite intensity on fish gills (p<0.05).
The mean number of Dactylogyrus sp. decreased from 50.60 ± 7.78 to 36.30 ± 5.52, from 69.20 ± 21.95 to 47.70 ± 14.71, and from 54.60 ± 7.04 to 35.40 ± 9.58 for peppermint, lemon, and tea tree essential oils, respectively (Fig. 5). The antiparasitic efficacies were calculated as 28.23 % for peppermint essential oil, 30.95 % for lemon essential oil, and 35.31 % for tea tree essential oil.
Fig. 5.
Dactylogyrus sp. mean intensity in carp gills before and after in vivo application of peppermint, lemon, and tea tree essential oils.
Discussion
Restriction or prohibition in many countries of traditional chemical therapies, concerning their harmful side effects on fish, humans, and the environment, made it necessary to find alternative therapeutics to combat parasitic diseases in fish. For this reason, there is an increasing amount of research on the potential applications of natural products and herbal substances (Tavares-Dias, 2018). Treatment potentials of various herbal products against different parasites in fish have been studied (Wang et al., 2011; Wu et al., 2011; Lu et al., 2012; Militz et al., 2013; Ramudu & Dash, 2013; Valladao et al., 2015; Tavares-Dias, 2018; Yavuzcan et al., 2019; Yildiz & Bekcan, 2020). However, studies on the antiparasitic effects of essential oils against monogeneans are limited for some fish species. Antiparasitic activity of peppermint, lemon, and tea tree essential oils was assessed in this research in vitro and in vivo against Dactylogyrus sp. on carp.
In the in vitro tests of our study, peppermint, lemon, and tea tree essential oils showed different levels of antiparasitic effect against Dactylogyrus sp. collected from carp. These effects were determined to be dose and duration dependent and to increase with rising essential oil concentration and exposure time. Similarly, the dose and time-dependent antiparasitic effect of peppermint essential oil were observed in Malheiros et al. (2016)'s study, where 80 mg/l, 160 mg/l, and 320 mg/l concentrations of peppermint essential oil were applied in vitro on Dawestrema spp. While 100 % of parasites were dead in 5 hours at the lowest concentration (80 mg/l), at the highest concentration (320 mg/l) 100 % mortality had been observed in 30 min. In another study, da Costa et al. (2017) demonstrated the dose and time-dependent antiparasitic effect of peppermint and tea tree essential oils for monogenean parasites (Anacanthorus penilabiatus and Mymarothecium viatorum) collected from pacu (Piaractus mesopotamicus). In vitro antiparasitic effects of peppermint and tea tree essential oils in our study against Dactylogyrus sp. are in line with the antiparasitic effects observed in the study of da Costa et al. (2017). Time and dose-dependent patterns of antiparasitic effects of peppermint and tea tree essential oil were also observed for Ciliata parasite, Ichthyophthirius multifiliis trophonts (Valladão et al., 2016). In in vitro experiments here, cumulative mortalities of the parasites reached 100 % after treatment with peppermint, lemon, and tea tree essential oils in approximately 15, 10, and 3 minutes, respectively. Thus, the essential oil with the highest anthelmintic effect against Dactylogyrus sp. has been tea tree essential oil and the one with the lowest effect has been peppermint essential oil.
Plant sources previously studied for their antiparasitic potential against Dactylogyrus sp. were Ginkgo biloba, Dioscorea zingiberensis (Jiang et al., 2014), ginger bulb (Zingiber officinale) and pomegranate peel (Punica granatum) (Phan et al., 2021). Following ginger bulb and pomegranate peel treatments, the death of Dactylogyrus was observed between 1 min to 9 min for the ginger bulb and between 1 min to 15 min for pomegranate peel. The results of Phan et al. (2021)'s study are similar to our study in general. Zoral et al. (2017) assessed in vitro and in vivo anthelmintic activity of Rosmarinus officinalis against Dactylogyrus minutus infections in C. carpio. It took 61.8 ± 5.6 min and 7.8 ± 1.4 min to kill 100 % of parasites when exposed to 100 g/L and 200 g/L aqueous rosemary extract solution, respectively. Thus, peppermint, lemon, and tea tree essential oils in our study appear to have higher antiparasitic effects against Dactylogyrus sp. than R. officinalis against D. minutus.
The antiparasitic efficacy of peppermint, lemon, and tea tree essential oils in in vivo tests with carp have been found as 28.23 %, 30.95 %, and 35.31 %, respectively. The fact that the antiparasitic effects of the tested essential oils were less in in vivo experiments than the antiparasitic effects in in vitro experiments, as assessed by EC50, can be due to the ability of parasites to be embedded into skin mucus (Trujillo-González et al., 2015; Yildiz & Bekcan, 2020). Hashimoto et al. (2016) observed that the monogenean parasite number (Cichlidogyrus spp.) in Nile tilapia decreased by 41.63 % after peppermint essential oil at 40 mg/l concentration with recurrent baths. Malheiros et al. (2016) investigated the antiparasitic effects of peppermint essential oil, in vitro and in vivo, against Monogeneans, Dawestrema cycloancistrium and Dawestrema cycloancistrioides, on Arapaima gigas gills. However, the efficacy of peppermint essential oil was low, resulting in no recommendation of its use. Ferreira et al. (2019) investigated the antiparasitic effect of peppermint essential oil in vivo on Piscinoodinium pillulare on Collosoma macropomum. The recommended concentration of 20 mg/l applied as short-term baths for 24-hour intervals showed antiparasitic efficacy of 42.97 %. Steverding et al. (2005) studied in vivo the antiparasitic effects of tea tree essential oil against Gyrodactylus spp. in Gasterosteus aculeatus, and the number of parasites was found to be reduced by 90 % after 2 days compared to the control group. Baldissera et al. (2017) observed that tea tree essential oil used as bath treatment at a concentration of 50 μl/l for 1 hour every day for 4 days on silver catfish (Rhamdia quelen) caused a 94.87 % reduction in I. multifiliis. The parasite (D. minutus) intensities in carp were significantly lower in Zoral et al. (2017)'s study when fish were exposed to 50 g/l of aqueous rosemary solution for 30 min. The exposure durations in our in vivo experiments were 2 min for tea tree essential oil and 5 min for peppermint and lemon essential oils. Lower values of antiparasitic efficacy of essential oils in our study in comparison to the previous studies can be explained by the short and single treatments with the essential oils tested in the present study.
In vitro and in vivo test results of our study presented the antiparasitic potency of lemon essential oil against Dactylogyrus sp. in carp. To the best of our knowledge, this is the first study to assess the antiparasitic potential of lemon essential against parasites in fish. The available studies on lemon essential oil were about its capacity to kill bacteria when applied as a feed supplement in different fish species such as Oreochromis mossambicus (Baba et al., 2016) and Labeo victorianus (Ngugi et al., 2017).
Some other herbal extracts were also tested against Monogeneans. For example, Ling et al. (2015) analyzed the antiparasitic potential of cinnamon essential oil against Dactylogyrus intermedius in goldfish under in vivo conditions, and anthelmintic efficacies up to 75 % were observed in higher concentrations. Jiang et al. (2014) demonstrated the synergistic anthelmintic effect of the combination of Ginkgo biloba and Dioscorea zingiberensis extracts against Dactylogyrus sp. infections in goldfish. Luo et al. (2016) studied synergistic combinations of 10 kinds of traditional Chinese medicines against Dactylogyrus sp. and found 11 synergistic anthelmintic pairs.
In our study, the bath treatments with peppermint, lemon, and tea tree essential oils resulted in the reduction of Dactylogyrus in the gills. The antiparasitic capacities were tea tree essential oil>lemon essential oil> and peppermint essential oil. The differences between the results obtained from in vitro experiments and those obtained from in vivo experiments can be explained by the fact that the response of biological systems in vivo may differ from what is observed in vitro due to the complexity of biological interactions such as the effects of the mucus layer in fish. It is important to note that the success of herbal essential oil treatments may not prove as successful in controlling parasites. The effectiveness of the treatment may vary depending on the parasite species, the life cycle of the parasite, and the purity or quality of the essential oil.
Conclusion
In this research, peppermint, lemon, and tea tree essential oils showed antiparasitic effects against Dactylogyrus sp. in the gills of carp. In in vitro tests, the antiparasitic effects were assessed to be time and dose-dependent. In vivo tests conducted on carp demonstrated peppermint, lemon, and tea tree essential oils have relatively reduced antiparasitic effects against Dactylogyrus sp. The fact that peppermint, lemon, and tea tree essential oils have the potential to cause a lower impact on Dactylogyrus sp. in carp gills.
