Efficacy of the Aqueous Extract of Azadirachta indica Against the Marine Parasitic Leech and Its Phytochemical Profiling

Zeylanicobdella arugamensis (Hirudinea), a marine parasitic leech, not only resulted in the mortality of the host fish (Groupers) but also caused economic losses. The current study aimed to elucidate the antiparasitic efficacy of the aqueous extract of the Azadirachta indica leaves against Z. arugamensis and to profile the composition via LC-Q Exactive HF Orbitrap mass spectrometry. Different concentrations (25, 50 and 100 mg/mL) of A. indica extract were prepared and tested on the parasitic leeches. The total mortality of leeches was noticed with an exposure to the A. indica aqueous extract. The average times required for the aqueous extract at concentrations of 25, 50 and 100 mg/mL to kill the leeches were 42.65 ± 9.20, 11.69 ± 1.11 and 6.45 ± 0.45 min, respectively, in a dose-dependent manner. The Orbitrap mass spectrometry analysis indicated the presence of five flavonoids (myricetin 3-O-galactoside, trifolin, isorhamnetin, quercetin and kaempferol), four aromatics (4-methoxy benzaldehyde, scopoletin, indole-3-acrylic acid and 2,4-quinolinediol), three phenolics (p-coumaric acid, ferulic acid and phloretin) and two terpenoids (pulegone and caryophyllene oxide). Thus, our study indicates that A. indica aqueous extract is a good source of metabolites with the potential to act as a biocontrol agent against the marine parasitic leech in aquaculture.

The infestation of the marine parasitic leech Zeylanicobdella arugamensis (Annelida: Hirudinea: Piscicolidae) ( Figure 1A,B) spread rapidly in Southeast Asian countries [3][4][5][6]. In Malaysia, the marine leech was first reported in a grouper (Epinephelus coioides) reared in floating cages with a 0.4-percent prevalence [3]; later on, the leeches were frequently isolated from various major species of marine fish reared in cages such as hybrid groupers (Epinephelus fuscoguttatus × E.lanceolatus), groupers (E. fuscoguttatus, E. lanceolatus), snappers (Lutjanus johnii, L. argentimaculatus and L. stellatus) and sea bass (Lates calcarifer) [3][4][5]. Further, around 60 percent of moribund sea bass fingerlings nurtured in cages were found , highly infested with Z. arugamensis, indicated by the arrow (The parasites were isolated and used for the current research), (C) = normal parasite (exposed to seawater only, untreated with extract or chemicals, no mortality was noticed), (D) = formalin-exposed parasite (exposed to 0.25% of formalin solution in seawater, resulted in mortality of the parasites) (E,F) = A. indica aqueous extract-exposed parasites (exposed to low, medium and the higher concentration of plant extract prepared in seawater, resulted in mortality of the parasites with a wrinkled body).
Extracts and essential oils from a natural product can act as herbicide, antimicrobial, anticancer and antiparasitic agents [11][12][13][14]. Azadirachta indica (neem plant) belongs to the family Meliaceae, commonly found in the Indo-Malayan region and other parts of the world [15,16]. Various parts of the plant, such as fruits, seeds, leaves, bark and roots, are a good source of bioactive compounds with antimicrobial, antipyretic, anti-inflammatory and antiparasitic properties [15][16][17]. The antimicrobial activity of A. indica leaves' solvent extracts against fish pathogenic bacteria (Aeromonas veronii, Aeromonas hydrophila, Acinetobacter junii, Acinetobacter tandoii, Acinetobacter spp. and Pseudomonas stutzer) isolated from Blackspot barb (Dawkinsia filamentosa) have been reported [18]. The aqueous extract of the plant leaf at a concentration of 150 mg/l has been reported to be effective against the pathogenic infection caused by Citrobacter freundii in tilapia [19]. The essential oils of A. indica have been reported to have an antimicrobial property against Enterococcus faecalis, Aerococcus viridans, Pseudomonas aeruginosa, Proteus mirabilis and Escherichia coli and an antiparasitic activity against caligid parasites on seabass [20,21]. The data have revealed that A. indica extracts are toxic to over 400 species of insect pests, some of which have developed resistance to chemically prepared pesticides [15,22]. The plant is also used for the removal of intestinal worms [15]. Currently, no data is available regarding the antiparasitic properties of A. indica aqueous extracts against the marine parasitic leech. Hence, this study aimed to elucidate the antiparasitic efficacy of the aqueous extract of A. indica against Z. arugamensis and to profile the composition using LC-Q Exactive HF Orbitrap mass spectrometry.

Physiochemical Parameters
The water quality parameters of the controls and plant-treated groups are provided in Table 1. Slight changes in the pH of the plant solutions were noticed when compared to the control groups, which could be due to the presence of metabolites with an acidic nature in the extract while the rest of the parameters remained constant.

Antiparasitic Properties of the Aqueous Extract of A. indica
The mortality time and percentage of the leeches treated with aqueous extracts of the plant were calculated (Table 2, Figure 1C-F). A total mortality of the leeches was noticed in negative control ( Figure 1D) and all the plant-treated groups ( Figure 1E-F), and the time taken was from 6.45 to 42.65 min. No mortality of the leeches was noticed in the normal control group ( Figure 1C) until 720 min (12 h).

Discussion
The marine leech Z. arugamensis is a notorious ectoparasite and distributed throughout the Indian Ocean [3][4][5]23,24]. For many years, fish farmers used toxic antiparasitic chemotherapeutics and insecticides to prevent or control parasitic infestations in aquaculture [25][26][27]. The accumulation of these chemical residues in water has caused impacts on the environment and may have lethal or sublethal effects on nontarget organisms [28]. For example, in Norway, when Neguvon and Nuvon pesticides were applied to control the copepod Lepeophtheirus salmonis (Caligidae) in salmon net-pen farming, there were harmful effects on several crustaceans near the farms [29]. However, plants are a good alternative and can be applied to control parasitic infestation as a natural remedy for sustainable aquaculture to avoid the negative effects of pesticides [10]. They show zero or less toxicity to the environment due to their biodegradability and are a great source of bioactive compounds [30,31].
In this study, we selected A. indica due to its antimicrobial, anti-inflammatory, antipyretic, insecticidal and acaricidal nature [15][16][17]22] and determined the antiparasitic potential of the aqueous extracts of A. indica leaves. The exposure of the aqueous extract resulted in the total mortality of leeches in a dose-dependent manner (Figure 1). In three different doses used, all leeches were killed in an average period of 6.45 ± 0.45 min (100 mg/mL), 11.69 ± 1.11 min (50 mg/mL) and 42.65 ± 9.20 min (25 mg/mL). Previous studies have shown the effect of plant extracts on different fish parasites. The methanol extract of Dillenia suffruticosa (Dilleniaceae, tropical shrub) was applied against Z. arugamensis at a concentration of 100 mg/mL, and it took 14.39 and 4.88 min to kill all the leeches [32]. However, our treatment using the aqueous extract of A. indica took less time than D. suffruticosa to kill all the leeches. Further, the exposure of the methanol extract and chromatographic fraction (fraction 3) of Nephrolepis biserrata (Nephrolepidaceae, perennial fern) was applied against Z. arugamensis at a concentration of 100 and 2.5 mg/mL and killed all the leeches in 4.88 and 1.92 min, respectively [33,34]. Similarly, the methanol extracts of Allium sativum (Amaryllidaceae, garlic) (600 µg/mL) were tested against the aquatic leech Limnatis nilotica (Hirudinidae) and took 144.55 min for total mortality [35]. The essential oil of A. indica was applied against a crustacean parasite Caligus (Caligidae) infestation on the Asian seabass Lates calcarifer (Latidae) and showed a 100% mortality of Caligus within 5760 min (96 h) at a rate of 10 ppm [21]. The extract of Artemisia annua (Asteraceae, aromatic herbaceous plant, Sweet Annie) was reported to be effective in 30 to 180 min against monogenean parasites of the cultured airbreathing catfish Heterobranchus longifilis at concentrations ranging from 50 to 200 mg/L [36]. Thus, in comparison to the abovementioned plant, the aqueous extract of A. indica resulted in the total mortality of leeches in less than 7 min at a higher concentration. Furthermore, we explained the principle behind the antiparasitic nature of the plant via the identification of the different responsible metabolites using Q Exactive HF Orbitrap mass spectrometry.

Chemicals
Formalin (37% aqueous formaldehyde solution) and sodium bicarbonates were obtained from Sigma, Leica, Microsystem, and Germany. Methanol (HPLC grade) was purchased from Merck (Darmstadt, Germany). LCMS-grade acetonitrile, water and formic acid were obtained from Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA). Regenerated cellulose syringe filters with a 0.22-µm pore size and 13-mm diameter were obtained from Thermo Scientific (Thermo Fisher Scientific, Waltham, MA, USA).

Plant collection
The leaves of the plant A. indica (Figure 2)  gambiense without affecting the viability of the host cell [46]. Kaempferol inhibited th growth of the parasite Entamoeba histolytica (Entamoebidae), responsible for amoebiasis by altering cytoskeleton proteins [47]. It is also well known for its antiproliferative [48 antidiabetic [49], antioxidative [50], anti-inflammatory [51] and anticancer properties [52 Thus, we believe that the metabolites found in the aqueous extracts of A. indica could b responsible for the antiparasitic effect. Hence, we suggest that a natural-based treatmen could be a viable alternative to chemicals and is effective for eco-friendly and sustainabl aquaculture.

Chemicals
Formalin (37% aqueous formaldehyde solution) and sodium bicarbonates were ob tained from Sigma, Leica, Microsystem, and Germany. Methanol (HPLC grade) was pur chased from Merck (Darmstadt, Germany). LCMS-grade acetonitrile, water and formi acid were obtained from Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA Regenerated cellulose syringe filters with a 0.22-µ m pore size and 13-mm diameter wer obtained from Thermo Scientific (Thermo Fisher Scientific, Waltham, MA, USA).

Plant collection
The leaves of the plant A. indica ( Figure 2

Extraction
The leaves of the plant were rinsed with distilled water and dried in an oven at 3 °C. The dried plant was ground separately in a heavy-duty grinder. About 100 g of th dry plant powder was boiled with distilled water for 10 min with a 1:10 ratio (sample t the amount of distilled water) using a stirring hot plate. The decoctions were remove 5 cm

Extraction
The leaves of the plant were rinsed with distilled water and dried in an oven at 37 • C. The dried plant was ground separately in a heavy-duty grinder. About 100 g of the dry plant powder was boiled with distilled water for 10 min with a 1:10 ratio (sample to the amount of distilled water) using a stirring hot plate. The decoctions were removed and allowed to cool at room temperature for 1 h. Further, the extracts were filtered using a strainer to remove coarse residues, and then the filtrate was filtered again using Whatman No. 1 filter paper. The pure filtrate was kept at −80 • C for 24 h and then lyophilized using a freeze drier.

Source of Marine Leech Z. arugamensis
The marine leeches Z. arugamensis (1-1.5 cm) were procured from the aquaculture facilities. An infested hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus) (15-340 g) (diameter: 15-20 cm) with marine leeches (Figure 1A,B) was placed in a small tank containing seawater from the cage, and the leeches were removed individually by hand. The leeches were transferred into a container containing filtered seawater and incubated at 27 • C for 24 h.

Antiparasitic Bioassay
Adult parasites were selected, divided into five groups, and each group was provided with six parasites in a Petri dish. Group 1 served as a negative control ( Figure 1C), treated with seawater only, and group 2 served as a positive control, treated with 0.25% formalin solution ( Figure 1D). In contrast, groups 3, 4 and 5 were challenged with 25, 50 and 100 mg/mL of the aqueous extract of A. indica, respectively ( Figure 1E,F). During the challenge, parasites' inactivity and death were recorded using a stopwatch.

Liquid Chromatography
Liquid chromatography (LC) was performed using the Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a Thermo Syncronis C18 column (2.1 mm × 100 mm × 1.7 µm; Thermo Fisher Scientific, Waltham, MA, USA), which was maintained at 55 • C at a flow rate of 450 µL/min during analysis. The mobile phases were composed of solvent A (water added with 0.1% formic acid) and solvent B (acetonitrile added with 0.1% of formic acid). The gradient elution program was initiated at 0.5% of solvent B for 1 min, then from 0.5% to 99.5% of solvent B for 15 min and maintained for 4 min. Later, the columns were conditioned as initial for 2 min before the next injection. The injection volume was set at 2 µL.

Data Acquisition
MS and MS/MS data were acquired using the Thermo Scientific Q Exactive HF Orbitrap mass spectrometry system (Thermo Fisher Scientific, Waltham, MA, USA), which was equipped with a heated electrospray ionization (HESI) probe. The data acquisition was set between an m/z of 100-1000 for MS and 200-1000 for MS/MS. The resolutions of the MS and MS/MS data were acquired at 60 k and 15 k, respectively. Positive and negative HESI were both deployed at 3.5 kV and 3.0 kV, respectively. The ion source conditions were set as follows: capillary temperature of 320 • C, sheath gas flow rate of 50, aux gas flow rate of 18, sweep gas flow rate of 0 and aux gas heater temperature of 300 • C. Calibrations were performed using Pierce LTQ ESI Positive Calibrations solution and Pierce LTQ ESI Negative Calibrations solution (Thermo Fisher Scientific, Waltham, MA, USA) before sample analysis.

Data Analysis
The acquired data were processed and analyzed using Thermo Scientific Compound Discoverer 3.0 software (Thermo Fisher Scientific, Waltham, MA, USA) with the default settings in order to perform compound identification. Briefly, the default workflow includes background subtraction with blank data, retention time alignment, feature detection, elemental composition determination, libraries matching and fragment ion search (FISh) scoring. The identification of compounds was primarily based on the matching of MS/MS data against mzCloud and mzVault databases. Unmatched signals were attempted with the ChemSpider database using MS data and supported with a FISh scoring above 50.

Statistical Analysis
Data analysis was carried out using the IBM SPSS Statistics 25 Window package (IBM, Armonk, NY, USA). Significant differences between groups were determined using a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. All results were presented as the mean ±standard error of the mean (S.E.). p values under 0.05 were regarded as significant.

Conclusions
In the present study, it was proven that the application of various concentrations of A. indica aqueous extract indicated a strong antiparasitic activity with 100% mortality against the marine parasitic leech Z. arugamensis in an average time ranging from 6.45 to 42.65 min. The Q Exactive HF Orbitrap mass spectrometry analysis indicated the presence of five flavonoids (myricetin 3-O-galactoside, trifolin, isorhamnetin, quercetin and kaempferol), four aromatics (4-methoxy benzaldehyde, scopoletin, indole-3-acrylic acid and 2,4-quinolinediol), three phenolics (p-coumaric acid, ferulic acid and phloretin) and two terpenoids (pulegone and caryophyllene oxide). Some of the compounds were reported to have strong antiparasitic properties. This demonstrates that A. indica aqueous extract has the potential to act as a biocontrol agent against Z. arugamensis infestation. The antiparasitic properties of the aqueous extract of A. indica could be due to the presence of the above-mentioned metabolites. However, further investigation of the purification and isolation of the pure metabolites responsible for the antiparasitic properties is vital.
Supplementary Materials: The following are available online, Figure S1: Structures of the identified compounds (1-42) from the extract of Azadirachta indica, compound names are given in Table 3, Figure