Antiparasitic potential of Nephrolepis biserrata methanol extract against the parasitic leech Zeylanicobdella arugamensis (Hirudinea) and LC-QTOF analysis

Marine leech Zeylanicobdella arugamensis (Piscicolidae), an economically important parasite is infesting predominantly cultured groupers, hybrid groupers and other fish in Southeast Asian countries. In this study, we tested the anti-parasitic potential of a medicinal plant Nephrolepis biserrata found in Sabah, East Malaysia against Z. arugamensis. Various concentrations of methanol extracts of the plant were tested experimentally against Z. arugamensis and disinfestation of the leech from its primary host hybrid groupers. The composition of methanol extract of N. biserrata was determined through LC-QTOF analysis. The significant anti-parasitic activity of 100% mortality of leeches was observed with the exposure of N. biserrata extracts. The average time to kill the leeches at concentrations of 25, 50 and 100 mg/ml was 25.11 ± 3.26, 11.91 ± 0.99, and 4.88 ± 0.50 min., respectively. Further, at various low concentrations of N. biserrata 2.5, 5 and 10 mg/ml, hybrid groupers were disinfested in an average time of 108.33 ± 12.65, 65.83 ± 9.70 and 29.16 ± 5.85 min., respectively. The tandem mass spectrometry data from LC-QTOF indicated some hits on useful bioactive compounds such as terpenoids (ivalin, isovelleral, brassinolide, and eschscholtzxanthin), flavonoids (alnustin, kaempferol 7,4′-dimethyl ether, and pachypodol), phenolics (piscidic acid, chlorogenic acid, and ankorine), and aromatic (3-hydroxycoumarin). Thus N. biserrata can act as a potential biocontrol agent.


Scientific Reports
| (2020) 10:22091 | https://doi.org/10.1038/s41598-020-79094-4 www.nature.com/scientificreports/ host by the pathogenic bacteria such as Vibrio alginolyticus which further leads to the mortality of the host in a very short period 7,9,10 . Thus, Z. arugamensis is a vital threat to the grouper and other species in the aquaculture industry. Toxic chemicals have been used for the control, especially formalin which are harmful to both fish and human beings [11][12][13][14] . In addition, some other chemicals such as copper sulfate, potassium permanganate, and hydrogen peroxide, have also been used, they could affect the water quality and other parameters including health of human beings through accumulation of toxic contents in fish. Hence, researchers and farmers are looking for alternative approaches including the medicinal plants for the eco-friendly treatments 13 . The natural products such as medicinal plants are possessing anti-inflammatory and antipathogenic properties with minimal toxicity level to fish. To minimize the consumption of toxic chemicals plant derived extracts can be used as a natural control agent against parasitic infestation due to the presence of antiparasitic phytochemical compounds such as alkaloids, glycosides, terpenoids etc [15][16][17] . Hence, research on the development of safe and natural agent for controlling the infestation rate of this species is critical.
Nephrolepis biserrata is a perennial fern, belongs to the family Nephrolepidaceae 18 , locally known as "Paku pedang" 19 . In addition to Malaysia, the plant is also distributed in other South-east Asian countries 20,21 . In this medicinal plant, phytochemical profiling via gas chromatography mass spectrometry (GCMS) and the hepatoprotective properties have been studied 22,23 . Besides, the plant possesses antibacterial and antifungal properties 24 . However, no data have so far been reported concerning the anti-parasitic properties of N. biserrata against Z. arugamensis. The main objectives of the present study were to evaluate the anti-parasitic activity of N. biserrata methanol extract against the leech Z. arugamensis, disinfestation of the hybrid groupers (Epinephelus fuscoguttatus x E.lanceolatus) and chemical composition by LC-QTOF.

Results
Anti-parasitic properties of N. biserrata. The mortality time of Z. arugamensis in formalin and extracttreated groups is indicated in Table 1. The plant treated groups showed the antiparasitic effect in a dose-dependent manner. The time taken for the mortality of the parasitic leeches was lesser in 100 mg/ml dose than 50 and 25 mg/ml of the methanol extract. No mortality was noticed in the normal control group throughout the 720 min observation. biserrata is shown in Table 2. The normal control group is free of leeches. The negative control group indicated no disinfestation of the leeches until 720 min. The plant treated groups indicated the effect in a dose-dependent Table 1. Mortality time of the leeches at different concentrations of methanol extract of N. biserrata. Each value represents the mean ± S.D of 6 parasitic leeches per group. a Significance at p < 0.05 compared with the normal control group. b Significance at p < 0.05 compared with the formalin treated group (0.25% v/v). c Significance at p < 0.05 compared with N. biserrata (25 mg/ml). d Significance at p < 0.05 compared with N. biserrata (50 mg/ml).

No. Group
Mortality time (min) Mean ± S. D Mortality (%)  Behaviour of hybrid groupers. Fishes in the normal control group showed normal swimming activity ( Fig. 2a) and in the negative control group they were weak (Fig. 2b). Fishes treated with 0.1% formalin showed extreme aggressive behaviour throughout the treatment. Around body areas of the formalin treated groups were found with wounds ( Fig. 2c, d) as compared to the plant treated groups. However, active swimming was noticed in the group exposed to 10 mg/ml of the plant extract for the first 1 to 5 min later, they were relaxed. The fishes exposed to 5 mg/ml of the extract showed active swimming for 1 to 2 min while no active swimming was noticed in the fishes treated with 2.5 mg/ml of the plant extract. In the end, all the fishes treated with plant extracts were relaxed (Fig. 2e, f).

Physicochemical parameters of leeches treated solutions.
The water quality parameters of the control groups and methanol extract groups solutions applied for the anti-parasitic assays are indicated in Table 3.  Physicochemical parameters of hybrid groupers treated solutions. The water quality parameters of the normal, negative and positive control group solutions and methanol extract solutions applied for the fish disinfestation are indicated in Table 4. All parameters determined remained constant. The pH of the methanol extract solutions was maintained above 7 with the addition of sodium bicarbonate (I mg/ml) to provide an alkaline environment to the fishes.

LC-QTOF analysis and metabolites identification.
A total of 77 compound's features were extracted and determined for their molecular formula by executing Molecular Feature Extraction (MFE) algorithm in Agilent MassHunter software. Among these 77 metabolites, 18 of them were successfully matched with the METLIN databases and previously reported in plant extract (Table 5; Fig. 3).

Discussion
Ectoparasitic leech (Z. arugamensis) provided a significant threat to the grouper aquaculture facilities in Malaysia 8,25 . To control the leech infestation, formalin and other chemicals have been utilized by fish farmers, which cannot provide a conducive environment for the eco-friendly aquaculture policy 14 . On the other hand, plant-based extracts can be used as a safe and natural treatment for the parasite infestations due to the presence of antiparasitic secondary metabolites 16 . In addition to this, the secondary metabolites present in the plant extract also play an important role in the enhancement of growth and immunity of cultured species. The olive oil leaf extract has been reported to boost the activation of intestinal digestive enzyme activity and the expression of genes related with growth in brain, liver, muscle and other vital tissues of common carp Cyprinus carpio 26 . In the Table 4. Water quality parameters of the solutions applied for the fish treatment.
No. In the present study, the anti-parasitic potential of the methanol extract of N. biserrata was almost 3 times faster than D. suffruticosa methanol extract 28 . In another study, the parasitic leech Piscicola geometra from the infested cobia fish exposed for 96 h to various dilutions of the extracts of Scutellaria baicalensis and Morinda citrifolia and obtained 100% and 80% mortalities, respectively 28 30 .

Water parameters Concentrations
Low and suitable concentrations such as 2.5, 5 and 10 mg/ml of the plant extracts were administered for fish disinfestation to avoid the fishes from stress. The disinfestation of fishes was obtained in a dose-dependent manner ( Table 2). Groups of infested fishes treated with 10 mg/ml of the methanol extract were disinfested in less than 30 min. Similar results were noticed in the group treated with 5 and 2.5 mg/ml of the extract in the period of 65 and 108 min., respectively. The infested fishes treated with 0.1% formalin took about 26 min. to get rid of the parasitic leeches. According to statistical analysis, no significant differences were noticed in the disinfestation time of the formalin (0.1%) and plant treated group (10 mg/ml). This showed that the treatment effect of the higher concentration of the methanol extract of N. biserrata (10 mg/ml) was the same as that of formalin (0.1%) (v/v). Our results are in agreement with the anti-parasitic activity of solvent extract (petroleum ether, chloroform, ethyl acetate, methanol) and aqueous extracts of plants, Radix angelicae pubescentis, Fructus bruceae, Caulis spatholobi, Semen aesculi, and Semen pharbitidis against the monogenean Dactylogyrus intermedius in goldfish 31 . Among the experimental extracts, the methanol and aqueous extracts of S. aesculi were noticed to be more effective after 48 h of exposure, followed by methanol extracts of Fructus bruceae, Radix angelicae pubescentis, Caulis spatholobi, and Semen pharbitidis 31 .
In the present study, for the comparison of the anti-parasitic effect of the plant extracts, we used formalin as a positive control, since it is vastly utilized for the removal of parasites in aquaculture facilities 14 . Besides, other chemicals such as trichlorfon, hydrogen peroxide and copper sulfate have also been used as an anti-parasitic agent 32 . In Malaysia, other drugs like benzalkonium chloride, acriflavine, malachite green, hypochlorite and poly-vinyl pyrrolidine are also utilized for the removal of parasites in aquaculture facilities 33 . These chemicals are harmful to fish and their consumers 14,33 . However, the methanol extract of N. biserrata could be obtained from a natural source with no known side effects 23 .
On the other hand, isovelleral was found significantly inhibited germination of plant-parasitic fungus Alternaria brassicicola O-264 isolates conidial at as low as 0.05 ppm of vapour concentration 37 . However, anti-parasitic properties of the detected bioactive compounds in N. biserrata have not been reported on marine parasites hence their efficacies are pending for discovery. The strong antiparasitic effect of the methanol extracts of N. biserrata could be due to the presence of the above-mentioned metabolites [34][35][36][37] .
Our study proved that administration of the methanol extract of N. biserrata showed a strong anti-parasitic activity with 100% mortality against the marine leech Z. arugamensis parasitic on hybrid groupers in Malaysia. The exposure of the leech-infested hybrid groupers to methanol extracts of N. biserrata at a higher concentration (10 mg/ml) also resulted in the removal of parasitic leeches in less than 30 min. The tandem mass spectrometry data from LC-QTOF indicated some hits on terpenoids, flavonoids, phenolics and aromatic. The antiparasitic properties of N. biserrata could be due to the presence of various bioactive compounds. Some of the compounds have already been reported with strong anti-parasitic properties. It indicates that N. biserrata has a strong potential to act as a biocontrol agent against leech infestation in hybrid groupers. However, further study on the purification and isolation of the pure bioactive compounds responsible for the antiparasitic properties is imperative.

Materials and methods
Chemicals. Formalin (37% aqueous formaldehyde solution) and sodium bicarbonates were purchased from Sigma, Leica, Microsystem, and Germany. HPLC grade methanol was obtained from Merck (Darmstadt, Germany). LCMS-grade acetonitrile was purchased from J. T. Baker (Philipsburg, NJ, USA). Deionized water was acquired using Milli-Q system (Merck, Darmstadt, Germany) at resistivity of > 18. Solvent extraction. The leaves and stem of the plant were washed with distilled water and oven-dried in an oven at 37 °C for 3 days. The dried plant was grounded to a fine powder using a mechanical grinder and stored in an airtight container. About 60 g of dry powder of the plant was extracted with HPLC grade methanol (300 ml) using an orbital shaker (25-30 °C) for 3 days. The supernatant was filtered through Whatman No. 41 filter paper by vacuum and the fresh solvent was added to the samples, which were extracted for another 3 days. The methanol residues were removed from the extract using a vacuum rotary evaporator (R-215, BUCHI, Switzerland). The sample was kept at − 80 °C for 24 h and then lyophilized using a freeze drier (Freezon 12, Labconco, USA). The freeze-dried sample (4.80 g) was then stored in the freezer for further studies.
Anti-parasitic bioassay. Parasitic leeches were collected from the aquaculture facilities of Universiti Malaysia Sabah. The leeches were identified based on their morphological features 10 . Adult leeches were selected, divided into 5 groups (6 leeches per group) and each group was provided with six leeches in a Petri dish.
During the challenge, mortality time was recorded using a stopwatch for 720 min 27 . The experiment was performed in triplicate.
Observation on the behaviour of leeches. The changes in the behaviour of leeches were observed visually after exposure to formalin and different concentrations of plant extract as compared to the normal control group.
Fish disinfestation. Hybrid groupers (Epinephelus fuscoguttatus x E.lanceolatus) (TL: 15-20 cm) were obtained from the hatchery facilities of University Malaysia Sabah. All animals were treated humanely and well maintained under standard ethical principles as per university regulations (UMS/IP7.5/M3/4-2012). All experimental protocols were approved by the University Malaysia Sabah committee. They were acclimatized for 2 weeks before the start of the experiment. Later, all fishes except normal control group were infested with leeches for one week. Further, they were divided into the following groups (6 fishes per group).
Group 1: Normal control, treated with seawater only, no leech infestation (Fig. 2a). Group 2: Negative control, infested with leeches and no treatment was applied (Fig. 2b) www.nature.com/scientificreports/ During the experiment, the time required for the complete removal of all the leeches (disinfestation time) from the treated fishes was recorded using a stopwatch.
Observation on the behaviour of hybrid groupers. The changes in the behaviour of the hybrid groupers were monitored after exposure to formalin and different concentrations of the plant extracts in comparison to the normal control group.

Liquid chromatography quadrupole time-of-flight (LC-QTOF) acquisition. The methanol extract
of N. biserrata was analyzed using an Agilent 1290 Infinity LC system coupled with an Agilent 6520 QTOF mass spectrometry system. A 2.0 µl sample was injected into a reversed-phase column, namely Agilent Zorbax Eclipse XDB-C18 (narrow bore, 2.1 mm × 150 mm × 3.5 µm; Agilent Technologies, Santa Clara, CA, USA). The column was maintained at 25 °C at a flow rate of 500µL/min during analysis. The mobile phases were composed of solvent A (H 2 O-0.1% HCOOH) and solvent B (acetonitrile-0.1% HCOOH). The gradient elution program was initiated at 5% of solvent B for 5 min, then from 5 to 100% solvent B in 15 min. and kept for 5 min. Later, the column was conditioned as initial for 5 min. prior to next injection.
The mass spectrometry (MS) signals were acquired as described by Ling et al. (2018) 38 with minor modifications. Briefly, voltage for positive electrospray ionization was changed to 4 kV, while the remaining settings for ion source were maintained. The MS was calibrated with Tuning Mix (Agilent Technologies, Santa Clara, CA, USA) before each batch analysis. Internal mass calibration standards, purine (m/z 121.0508) and hexakis-(1H, 1H, 3H-tetrafluoropropoxy)-phosphazine (m/z 922.0097), were introduced throughout the runs for automated mass correction 39 . Identification of metabolites. Automated tandem mass spectrometry (auto-MSMS) was employed for metabolites matching, where precursor ions were selected consistently and fragmented throughout the data acquisition. Briefly, two major precursor ions were selected from each MS scan for fragmentation with nitrogen gas at a collision energy of 30 eV. Data acquisition for product ions was set between an m/z of 50 and 1500. Acquired data were processed and identified using Agilent MassHunter Qualitative Workflows software (version B.08.00), which is equipped with MassHunter METLIN Metabolite PCD/PCDL (Personal Compound Database and Personal Compound Database and Library).

Statistical analysis.
Data analysis was carried out using IBM SPSS Statistics 25 Window package (IBM, Armonk, NY, US). Significant differences between groups were investigated using one-way analysis of variance (ANOVA) followed by Tukey's test. All data points were shown as mean ± standard deviation (S.D.). p values < 0.05 were viewed as significant 27 .