Evaluation of Nematocidal Action against Caenorhabditis elegans of Essential Oil of Flesh Fingered Citron and Its Mechanism

Essential oils from flesh fingered citrons were obtained by mechanical pressing extraction under optimal conditions. -irty-three components, representing 78.25% of the total oil, were identified by gas chromatography-mass spectrometry (GC-MS). In this study, we explored the toxicity of the essential oil of flesh fingered citrons and the associated regulatory mechanism using Caenorhabditis elegans as a model organism.-emortality experiment showed that the LD50 value is 1.48mg/ml. Oxidative stress experiments showed that essential oils of flesh fingered citrons can reduce the antioxidation of nematodes and can shorten their lifespan. In survival rate experiments for verification, d-limonene had a nematocidal effect by destroying the waterproof protective layer on the surface of the worm. β-Pinene can interfere with the normal growth of nematodes according to behavioral experiments, and reproduction experiments showed that α-pinene has very strong nematocidal properties. c-Terpinene also has good nematocidal activity. -e results demonstrated that the essential oil of flesh fingered citron has great potential to act as a new nematicide.


Introduction
Controlling insect pests and nematodes by synthetic nematicides is expensive and environmentally harmful. e search for naturally occurring, cheaper, and more environmentally friendly biocontrol agents is ongoing throughout the world [1]. Currently, plant chemicals are being increasingly used as effective botanical nematicides in the form of extracts and essential oils because synthetic nematicides cause many negative effects on the environment [2]. erefore, we have explored natural plant sources for pest management agents. Due to the infrequent presence and inconsistent efficacy of synthetic pesticides, plant nematicides may be suitable alternatives to disrupt the physiological processes of target nematodes in addition to direct mortality [3,4]. Because of their nematocidal toxicity, essential oils are considered a possible alternative [5]. ese oils have been found to have significantly negative impacts on nematode survival, reproduction, development, behavior, and metabolic pathways [6,7] and have been studied as nematocidal, larvicidal, and repellent agents [8].
Essential oils are plant-based economically important secondary metabolites [9]. Citrus essential oils are extracted from citrus peels [10] that produce characteristic odors and have been widely applied to the food (beverage, sweets, and so on), cosmetics, and pharmaceutical industries mainly because of their fragrance and flavor [11]. e essential oils of the flesh fingered citron (Citrus medica cv. sarcodactylis) are used as an expectorant to treat cough, asthma, and inflammation, which is generally produced by mechanical extraction like all citrus oils [12]. Flesh fingered citron essential oil can be divided into two fractions, with the volatile fraction (93%-96%) containing monoterpenes and sesquiterpenes, such as d-limonene, α-and β-pinene, c-terpinene [13], and other oxygenated components (4%-7%). It is an essential oil of intermediate to low odor and flavor intensity [14]. Terpenoids that come from this essential oil are important nematocidal substances and have strong biological activity. us, this essential oil has been used in agricultural nematicide research. Different components like monoterpenoids (e.g., α-pinene) are responsible for nematocidal properties and physiological disruptions of nematodes [15,16].
ese compounds can be used as antifeedants, digestion and metabolism breakers, acetylcholinesterase inhibitors, and cell death inducers by acting as oxidizing agents, which can cause ionic leakage of cells and degeneration of mitochondria and proteins [17][18][19][20][21][22]. Limonene, a monocyclic monoterpene, is famous for its citrus-like olfactory properties [23]. Limonene is also an important precursor for many herbal and pharmaceutical compounds such as perillyl alcohol, carvone, and menthol [24,25]. D-Limonene is the most widely used isomer in spices, medicines, and pesticides [26]. As a nematicide, it can break the waterproof protective layer of the worm's body surface and has strong penetrating properties so that worms die by suffocation, reducing their survival rate.
Many studies have employed the nematode Caenorhabditis elegans as an animal model to study the effects of essential oils [27]. C. elegans is also used as an epidemiological model for stress and aging because of its small size, relatively short lifecycle, and the clear impact of genetic and environmental factors that affect the lifespan [28].
In this study, we used C. elegans as an in vivo model to determine the mortality effect of essential oils. We examined the mechanism of action of essential oils and the main individual components on C. elegans mortality and tested our hypotheses through a series of experiments on oxidative stress, lifespan, reproduction, and survival rate.

Plant Materials and C. elegans.
Mature fruits of the flesh fingered citron were harvested in November 2017 from a local orchard in Jinhua City, Zhejiang Province, P.R. China. Wild-type Caenorhabditis elegans (N2) and food E. coli OP50 strains were obtained from the Caenorhabditis Genetics Center.

Essential Oil Preparation.
e mechanical pressing method is suitable for the extraction of easily burnt raw materials such as citrus and lemon because it is easy to maintain the structure and function of essential oils. Flesh fingered citron fruits were first screened and peeled, saturated in 3% (w/v) calcium chloride or calcium hydroxide for a specified time (4 h) to remove pectin, and then cleaned and leached with distilled water. e soaked flavedo (50 g) was weighed and turned into juice using a juice extractor. e juice was supplemented with sodium chloride (0.25%) and stirred until the sodium chloride completely dissolved. After the solution was separated into layers, the oil-water mixed liquid obtained after filtering was centrifuged, and the upper oil layer of the mixture was separated and dried with anhydrous sodium sulfate [29]. e extraction rate of essential oils was about 0.3%. e purification was carried out using a molecular distillation method carried out by a laboratory rubbed molecular distillation device (Pope, USA). At 45°C melted in the water bath, essential oil was drawn into a feed bottle and degassed using a short-path evaporator. e distillation temperature, distillation pressure, wiping roller speed, feed flow rate, and cooling water temperature were set to 70°C, 50 Pa, 200 rpm, 4 mL/min, and 10°C, respectively. en, the feeding was turned on, and the degassed feed liquid immediately became a very thin film and spread onto the evaporation surface. A different number of volatile components evaporated as the liquid flowed down the wall through the heating zone. More volatile components concentrated onto the closely positioned internal condensing surface and became the distillates, while the less volatile components flowed down along the cylinder and became the residues. After distillation, the distillate and residue fractions were weighed and transferred to the containers. ey were stored at 4°C until analysis [30]. e purification rate of essential oils was about 85%.

GC and GC-MS Analysis.
A fused silica capillary column (HP-5MS) was used for GC analysis with a Hewlett-Packard 6890 gas chromatograph (5% phenyl-methylsiloxane, 60 m × 0.25 mm, film thickness 0.25 μm, Agilent Technologies, USA). e oven temperature was set to rise from an initial temperature of 50-100°C at a rate of 10°C/min and held there for 10 min and then raised to 140°C at a rate of 3°C/min. It was kept at 140°C for 10 min and finally heated to 230°C at a rate of 2°C/min and held there for 10 min. e split injection mode with a split ratio of 1 : 10 was employed. Quantification of components in the essential oil was obtained based on the GC peak areas using both the internal standard method and relative response factors. GC/MS analysis was performed on a Hewlett-Packard 6890 gas chromatograph coupled with a Hewlett-Packard mass-selective detector 5973N quadrupole mass spectrometer (MS) (Agilent Technologies, USA). All GC experimental parameters were the same as described for GC-FID analysis. e MS was operated in the electron impact ionization (EI) mode with an ionization energy of 70 eV. e temperatures of the MS source and MS quadrupole were 280 and 150°C, respectively. e constituents of the essential oil were identified based on computer matching with the Wiley/NIST library and comparisons of retention indices with those reported in the literature [31].

C. elegans Strains, Handling, and Mortality.
e nematodes used in this study were Bristol wild-type N2 [32]. C. elegans was grown and assayed on NGM agar plates carrying E. coli OP50 at 20°C. e phytochemicals were dissolved in 0.1% DMSO. All chemicals in the NGM plate and liquid are expressed in the final concentrations.
To investigate the nematocidal effect of the flesh fingered citron essential oil, a survival assay was deployed. In this assay, the worms were treated with essential oils at varying concentrations of 0, 0.5, 1, 1.5, and 2 mg/ml. C. elegans mortality was determined after 24 h exposure, and LD 50 was calculated [33].

Oxidative Stress Resistance Assays for C. elegans.
Oxidative stress assays were performed essentially as previously described [34]. Wild-type N2 C. elegans was synchronized to the L4 period and pretreated with 0, 0.5, 1, 1.5, and 2 mg/ml essential oil and 0.1% DMSO as a solvent control for 48 h, then exposed to K 2 Cr 2 O 7 (8 μ l, 10 mmol/L), and finally cultured for 48 h. Worms were observed every 4 h, and their survival was determined by the touch-provoked movement [35]. At least three independent biological replications were performed.

Lifespan
Assays on C. elegans. L4 larval C. elegans (wild-type N2) were transferred to 96-well plates with S-medium to inhibit progeny development in the presence of 0, 0.5, 1, 1.5, and 2 mg/ml essential oil of flesh fingered citron dissolved in 0.1% DMSO. We evaluated whether the essential oil influenced the lifespan of C. elegans by comparing the lifespans of untreated (control) and essential oiltreated wild-type N2 C. elegans. Live and dead C. elegans was counted every other day (from the first day of adulthood) until all C. elegans had died as determined by the touchprovoked movement. At least three independent biological replications were performed.

Survival Rate of C. elegans.
As a verification experiment, L1 stage larvae were treated with the essential oil solution for 3 days to the L4 stage and exposed to air for half an hour, and then the survival rate was determined. We performed at least three independent biological replications.

Behavioral Experiments on C. elegans. Body bends and
head swings indicate alternating muscle contractions. Synchronized L4 adults were transferred to an empty NGM board and allowed to be free for at least 1 min. e number of body flexions within 20 sec and the number of head swings with a drop of distilled water within 1 min were determined. We performed at least three independent biological replications.

Reproductive Assays of C. elegans.
e effect of essential oil on fecundity was assessed using age-synchronized N2 wild-type worms [36]. e total progeny yield of each worm after exposure to essential oil (0, 0.5, 1, 1.5, 2 mg/ml) was assessed according to standard protocols. Essential oiltreated/untreated L4 larvae were transferred to fresh treatment/control plates until reproduction terminated.
ree days later, the number of nematodes in the two plates was determined. We performed at least three independent biological replications.

Mortality Rate Inflicted by Four Key Components in Essential Oil.
e experiments were similar to those in Section 2.5. Four major components, D-limonene, c-terpinene, α-pinene, and β-pinene (5 mM each), were used to treat C. elegans. At least three independent biological replications were performed using at least 100 C. elegans strains.

Statistical Analysis.
For the oxidative stress assays and lifespan assays, we used GraphPad Prism (GraphPad Software). Survival curves resulting in p values <0.05 were considered significantly different relative to untreated controls. Statistics were analyzed using SPSS Statistics Software (SPSS). Results are expressed as the mean-± standard error of the mean (SEM). Univariate analysis of variance was performed using the ANOVA algorithm, while the LSD test was used for minimally significant difference analysis. Post hoc tests to determine the significance and interaction between the factors of the lifespan of C. elegans were performed with a two-factor variation analysis (twoway ANOVA). Differences were considered significant at the P < 0.05, P < 0.01, and P < 0.001 levels [35].

GC-MS.
Qualitative and quantitative analytical results of the chemical composition of essential oil of flesh fingered citron by GC/MS are listed in Table 1. Quantification of analytes was carried out by peak area normalization and expressed in terms of the percentage of the total peak area to facilitate comparison with data reported in the literature. As shown in the table, 33 compounds representing 78.25% of the total peak area were identified. e essential oil consisted mainly of monoterpene hydrocarbons, and the main ones were d-limonene (43.39%), c-terpinene (18.57%), α-pinene (2.88%), β-pinene (2.69%), β-myrcene (1.77%), (Z)-β-ocimene (1.46%), and α-thujene (1.31%). e components of EO in the present study had a composition similar to that in the previous reports about other species of citrus fruits [37,38].

Essential Oils of Flesh Fingered Citron Kill Wild-Type C. elegans.
e lethal doses (LD 50 ) were used to compare the effects of the essential oil exposure in contact toxicity assays [15]. In residual toxicity bioassays, mortality differences were found according to the concentration. e mortality rate of the essential oil of flesh fingered citron to C. elegans is Journal of Chemistry linear for each dose concentration range (Figure 1). When the concentration range of the essential oil is 1.0-1.5 mg/ml, the equation can be obtained from the linear relationship: y � 46x − 18, so the LD 50 value is 1.48 mg/ml.

Essential Oil Affects Oxidative Stress Resistance of C.
elegans. It has been widely reported that many kinds of citrus essential oils from natural plants exhibit antioxidant, antiradical, and antimicrobial properties [34]. K 2 Cr 2 O 7 is an oxidative stress inducer because of its heavy metal oxidant that produces an intracellular oxidation-reduction reaction with O 2 production and is commonly used to generate intracellular oxidation in C. elegans [39]. Pretreatment with different concentrations of the essential oil of flesh fingered citron in 0.1% dimethyl sulfoxide (DMSO) had a nematocidal effect on C. elegans via oxidative stress ( Figure 2). Essential oils contain a variety of compounds that may unleash different biological activities. Moreover, higher concentrations of the extract may be toxic, and the higher the concentration, the greater the toxicity, thus reducing the antioxidation effect of C. elegans.

Essential Oil of Flesh Finger Citron Affects the Lifespan of Wild-Type C. elegans.
e C. elegans model system was used to assess the effects of essential oils on aging. e increase in free radical production and the destruction of the redox balance lead to oxidative stress, causing aging and agerelated diseases [40]. Senescence is well illustrated in C. elegans and shares homologous mechanisms with primates and humans [41][42][43]. e experimental results (Figure 3) show that the higher the concentration of essential oil, the more obvious the nematocidal effect, and the lower the concentration of essential oil, the longer the survival of C. elegans. is indicates that the essential oil of flesh fingered citron can reduce the lifespan of C. elegans.

Survival Rate.
e formation of allergenic oxidation products like D-limonene oxide and dD-limonene 2-hydroperoxide has been reported [44] for oxidized citrus oil (rich in d-limonene), a frequent skin sensitizer. D-Limonene can break the waterproof protective layer of the worm's body surface and has strong penetrating properties so that worms die by suffocation. It can be seen from the graph (Figure 4) of the survival rate of C. elegans that the presence of essential oils of flesh fingered citron reduces the survival rate of nematodes.

Slowed
Behavioral Activity in C. elegans. Terpenes are the largest group of phytochemicals resulting from secondary metabolism [45]. Some components (e.g., β-pinene)   Journal of Chemistry are highly toxic and interfere with the normal growth of C. elegans and affect behavior. It can be seen from the results ( Table 2) that different concentrations of essential oil have a strong inhibitory effect on the number of body bends of nematodes and reduce the number of head swings. e essential oil of flesh fingered citron therefore affects the behavior of nematodes in addition to its nematocidal action.

Reproduction Rate Reduced in C. elegans.
As an essential oil component, c-terpinene has good insecticidal activity [46] and is highly toxic. α-Pinene is a strong repellant [47], has nematocidal characteristics, and induces physiological changes in some nematodes, and through the production of oxidizing compounds, it acts as an antifeedant, digestive and metabolic disruptor, acetylcholinesterase inhibitor, and cell death inducer, leading to the ionic leakage of cells and the degeneration of mitochondria and proteins [6]. It can be seen from the fertility rate ( Figure 5) that because of reduced synthesis of DNA, the reproduction of nematodes treated by the essential oil was lowered.  0.5mg/ml 1mg/ml 1.5mg/ml 2mg/ml * * * * * * * * * * * * Figure 4: Effects of essential oils on survival rates in C. elegans. Results show at least three independent biological replicates. Error bars represent the standard error, and differences compared to the control (0 mg/ml, 0.1% DMSO) were considered significant at * p < 0.05, * * p < 0.01, and * * * p < 0.001 by one-way ANOVA and the LSD post hoc test. At least three independent biological replications were performed, and statistical significance of the differences between curves was demonstrated by a log-rank test using Kaplan−Meier survival analysis. Error bars represent the standard error, and differences compared to the solvent (0 mg/ml, 0.1% DMSO) were considered significant at * p < 0.05, * * p < 0.01, and * * * p < 0.001 by one-way ANOVA and the LSD post hoc test.

Effect on Mortality of Four Key Components in the Essential Oil.
e structural formulae of four components are shown in Figure 6(a). e survival rate of nematodes exposed to d-limonene was 30% at 24 h; it was 34% for c-terpinene, 11% for α-pinene, and 9% for β-pinene ( Figure 6(b)). e survival rate of nematodes exposed to  Reproductive quantity 0 0.5mg/ml 1mg/ml 1.5mg/ml 2mg/ml The first day The second day Total * * * * * * * * * * * * * * * * * * Figure 5: Effects of essential oils on the reproduction rate in C. elegans. Results include at least three independent biological replicates. Error bars represent the standard error, and differences compared to the control (for each column of 0 mg/ml, 0.1% DMSO) were considered significant at * p < 0.05, * * p < 0.01, and * * * p < 0.001 by one-way ANOVA and the LSD post hoc test. α-pinene was 23% and that of β-pinene was 15% at 18 h. erefore, the components of the essential oil of flesh fingered citron have obvious nematocidal effects.

Conclusions
Plant essential oils and extracts directly affect nematode survival [48]. Essential oil extracted from the flesh fingered citron can be used as a plant-derived nematicide to kill C. elegans. At a dose of 2 mg/ml (about 0.2%, v/v), the mortality rate reached 88%. ese concentrations are much lower than the concentrations of insecticidal and nematocidal essential oils in the literature studies [49,50], and the LD 50 value was 1.48 mg/ml. Furthermore, oxidative stress experiments showed that the essential oil can shorten the survival time of nematodes by reducing the antioxidant activity of C. elegans. Essential oils can also kill C. elegans by reducing its lifespan, in addition to affecting behaviors such as body bends and head swings and lowering the reproductive rate. Finally, four isolated components had a significant mortality effect on the nematodes, indicating that these four terpenes in the essential oil are responsible for its effects. Monoterpenes are the main ingredients and the key nematicides. is article uses C. elegans as a biological model to kill nematodes in the realistic laboratory, which lays an experimental basis for the subsequent development of new natural pesticides. It has a very important theoretical significance and application value. is essential oil is a candidate for partial or complete replacement of current chemically synthesized pesticides to enhance sustainability.

Data Availability
e numerical data used to support the findings of this study are included within the article.

Additional Points
Highlights. (1) A kind of plant-derived nematicide is proposed. (2) e lethal mechanism of flesh fingered citron essential oil on nematodes is studied. (3) e nematocidal effect is excellent which also can replace chemically synthesized nematicides, which is sustainable and environmentally friendly.

Conflicts of Interest
e authors declare that they have no conflicts of interest.