In vitro Acaricidal and Repellent Effects of Amomum subulatum Essential Oil Against Hyalomma Ticks

Ticks are the main reservoirs of tick-borne pathogens of medical and veterinary concern. Tick- and tick-borne diseases prevail all over the world, typically in tropical and sub-tropical areas and affect almost 30% of the total cattle population (Basit et al., 2022). Among devastating parasitic infections of livestock, tick- and tick-borne diseases are ranked after mosquitoes and are considered as the most typical arthropod-borne diseases of livestock, humans, and companion animals (Abdelbaset et al., 2022). These ticks and tick-borne infections have doubled in the last two decades (Sanchez-Vicente et al., 2019). Ticks cause direct and indirect losses to the livestock e.g., they induce fever, anemia, irritation leads to chronic stress, immunodepression, hide damage, poor feeding leading to lethargic condition, weight loss, decreased milk production, and in females increased calving interval (Kasaija et al., 2021). Multiple species of ticks infect humans and animals, but the Hyalomma spp. are among the most common ticks infesting and serving as a vector for zoonotic agents (Rjeibi et al., 2022). Hyalomma spp. are among the most considered species of arachnids to be controlled by the farmers and researchers (Valcárcel et al., 2023).

Various acaricides like synthetic pyrethroids organophosphates, carbamates, and organochlorines are being used to control these ticks but their frequent use is resulting in the development of resistant tick species (Selles et al., 2021). Acaricidal resistance has threatened the welfare of livestock farmers globally because it led to decreased productivity and the emergence of infections (Ahmed et al., 2022; Betelhem et al., 2022; Saeed and Alkheraije, 2023; Sikander et al., 2023). Vaccines for the control of ticks have also been developed and being used commercially, but their efficacy is questionable, moreover they have limited value for the control of ticks (Bonnet et al., 2022). The vaccines in the future, if work successfully, cannot replace the therapeutic control measures. In this scenario, the need for alternatives is at prime for the control of ticks (Semenza et al., 2022).

Multiple alternative strategies are being suggested by the scientists, including metallic nanoparticles, entomophagous fungi, botanical products etc. (Raheel et al., 2021; Srisanyong et al., 2021; Hussain et al., 2022). Botanicals have always been a priority for the researchers to use as control of parasitic diseases (Abbas et al., 2020; Mubashir et al., 2022; Tanda et al., 2022). Multiple forms of botanicals i.e., whole plants, plant parts, extracts and essential oils etc. are in consideration (Sobhy et al., 2021; Bangulzai et al., 2022). The essential oils have the most promising importance among the botanicals because of the active compounds present in them (Radwan et al., 2022; Saeed and Alsayeqh, 2023). The essential oils of various plants have been proven to contain multiple medicinal properties (Ali et al., 2022; Bangulzai et al., 2022; Salman and Imran, 2022). Multiple essential oils have been tested for their acaricidal activities (Shezryna et al., 2020; Chaimanee et al., 2021; El-Sayed et al., 2022).

Amomum subulatum is commonly known as the Indian black cardamom or large cardamom (Saeed et al., 2023). It is commonly found in the sub-continent region, and it is famous for its medicinal and culinary usage (Joshi and Piya, 2019). Essential oil of A. subulatum is a dark liquid having a pungent smell (Kumar et al., 2022). A. subulatum has been found to be antiparasitic and acaricidal in multiple experiments (Al-Hoshani et al., 2023a). This experiment was designed to evaluate the acaricidal activities of essential oil of A. subulatum against Hyalomma spp. because of its medicinal properties of A. subulatum. Researchers also checked its effects against arthropods but not particularly against Hyalomma ticks.

Hyalomma anatolicum (H. anatolicum), Hyalomma dromedarii (H. dromedarii), and Hyalomma marginatum (H. marginatum) were collected carefully from various parts of Faisalabad by dragging technique, as described by Falco and Fish (1992). The ticks were kept in perforated plastic bottles with labels to allow for air and moisture exchange. The ticks were then brought to the Chemotherapy Laboratory, Department of Parasitology, University of Agriculture, Faisalabad, Pakistan. An identification guidebook was used for the identification of ticks based on morphological traits (Estrada-Peña et al., 2017).

GC-FID technique was performed for the evaluation of the composition of A. subulatum. This technique was performed at Central Hi-tech Laboratory, University of Agriculture Faisalabad, Pakistan, using a GC-17 SHIMADZU® spectrophotometer. The columns were adjusted at DB WEX 30M 0.25, and nitrogen was used as a mobile phase at the flow rate of 20mL/min. Adjustment of temperature was done at 90°C for 120 sec, then at 180°C for the same time and then at 240°C for 180 sec. The observations were recorded and maintained accordingly. The compositions verified the essential oil’s purity, and the active compounds present in them (Zhao et al., 2021).

In vitro acaricidal activity of A. subulatum essential oils was performed by preparing different dilutions. For this purpose, 0.31, 0.62, 1.25, 2.50, and 5% dilutions of A. subulatum essential oil were prepared with absolute alcohol (volume/volume; v/v), for adult immersion, larval immersion, egg hatch and tick repellency assays. For the adult immersion, larval immersion and egg hatch tests, absolute alcohol was maintained as negative control and 0.1% cypermethrin (Alphakill® by Agrichem Pharmauticlas, China) was used as a positive control. While for the acaricidal assay, N, N-diethyl-meta-toluamide (Pakistan Chemicals®; DEET) was used as positive control for the repellent assay and absolute alcohol remained as negative control. All the groups in all the test were replicated thrice for each test.

The Drummond et al. (1973) methodology was adhered to for adult mortality. Ten female ticks were initially weighed and immersed in various A concentration in accordance with this methodology five min with essential oil of subulatum. Ticks were dipped, then put in petri dishes with moist filter sheets and in a biological oxygen demand incubator set to maintain a temperature of 27°C and a humidity of 90% for a full day. Subsequently, the adult mortality was determined by counting both dead and live female ticks. After receiving dilutions, live female ticks were placed in perforated Eppendorf tubes and incubated for 20 days at 90% humidity and 27°C. Tick females deposited their eggs in Eppendorf tubes. To calculate reproductive parameters, egg mass and the residual weight of each individual female tick were recorded. For every concentration, the experiment was run three times, with a positive and a negative control. In this test, the following parameters were noted:

Adult mortality was performed according to Drummond et al. (1973) guideline sand calculated according to the following formula:

RI (%) was calculated Toro-Ortiz et al. (1997) using the following formula:

NI% was calculated using the following formula:

IO% was calculated by using the following formula:

Where IO and PE represented the inhibition of oviposition and product effectiveness, respectively.

The larval immersion test was used to calculate the effectiveness of A. subulatum essential oil on the larvae of Hyalomma spp. modified by Rosado-Aguilar et al. (2010). Tick larvae (7–14 days old) were utilized in this investigation. All the treatments and control groups were added into micro-centrifuge tube and 100 larvae were kept in each tube. After that, each tube was closed tightly and shaked vigorously for 5 sec and then gently for 5 min. Tubes were then opened and all larvae were shifted over filter paper to dry. These filter papers were then placed in an incubator for 24 h at 27°C and 90% relative humidity. Data were collected by counting dead and alive larvae with the help of a stereomicroscope (Rosado-Aguilar et al., 2010).

In glass tubes, 300 Hyalomma eggs were submerged for 5 min in 5mL of A. subulatum essential oil. After decanting the solutions, the tubes were sealed with cotton plugs and incubated for roughly 14 days at 27 to 28°C and a relative humidity of 70 to 80%, until the eggs began to hatch. Ethanol was utilized as negative control and cypermethrin was used as positive control. There were three replications of each treatment. Larval hatchability and inhibition of larval hatchability percentages were recorded (Kaaya et al., 1996).

Where Hc and Ht represented the hatchability in control and treated groups, respectively.

The vertical migratory kind of behavior of adult ticks, which was created and redesigned by Tabari et al. (2020), was utilized to evaluate repellant activity. For this purpose, ten ticks were observed to check the repellency behavior of Hyalomma ticks against A. subulatum essential oil with absolute alcohol as negative control and DEET as a positive control. The whole experiment was conducted at 27°C and 90% relative humidity. After one hour, the number of ticks above and below the filter paper strips were counted for 15 min and the percentage repellency was calculated.

Statistical analysis was done through one-way analysis of variance (ANOVA) and Tukey’s test by Minitab software while Probit Analysis by IBM SPSS software by keeping 95% confidence level and considering the results to be non-significant when P>0.05.

In the essential oil of A. subulatum, 13 chemical compounds were found among which the Limonene was present in greater concentration. The compounds are represented in Table 1; the peeks in the GC-FID assay are given in Figure 1.

Table 1: Phytochemical composition of A. subulatum essential oil through gas chromatography-flame ionization detection.

An adult immersion test was used to assess A. subulatum’s acaricidal activity against adult Hyalomma ticks at five different concentrations (0.31, 0.62, 1.25, 2.50, and 5%). The outcome showed that 5% of A. subulatum exhibited a significant (p<0.05) acaricidal effects against adult ticks as compared to negative control. Table 2 shows the lethal concentrations, or LC50 and LC90, that have also been found to be 2.80 and 21.11%, respectively. Probit analysis with a regression equation revealed a positive correlation between the concentration of A. subulatum essential oil and adult mortality. This showed that the concentration of essential oils increased together with an increase in adult mortality (Figure 2).

Table 2: Effect of various concentrations of A. subulatum essential oil against adults and larval stages.

C1, A. subulatum oil 0.31%; C2, A. subulatum oil 0.625%; C3, A. subulatum oil 1.5%; C4, A. subulatum oil 2.5%; C5, A. subulatum oil 5%; CN, Control Negative; CP, Control Positive. Mean±SD along with the same superscripts have a non-significant difference (p>0.05) from each other.

The larvicidal effect of A. subulatum essential oil at five different concentrations (0.31, 0.62, 1.25, 2.50, and 5%) was evaluated against the larvae of Hyalomma ticks by using a larval immersion test. Result revealed that 5% concentration of A. subulatum showed a significant (p<0.05) larvicidal effect against larvae of Hyalomma ticks. LC50 and LC90 values were also calculated which were 2.08 and 19.75%, respectively (Table 2). A. subulatum essential oil concentration and larval mortality were found to be positively correlated by probit analysis using a regression equation. This indicated that as essential oil concentration increased, adult mortality also did so, as illustrated in Figure 3.

There is evidence showing that the essential oil extracted from A. subulatum can decrease the Hyalomma tick’s reproductive capabilities. At 5% concentration, A. subulatum essential oil reduced Hyalomma tick oviposition by 64.88%. Additionally, it induced larval hatching at 38% and inhibited larval hatching at 62%. As the concentration of A. subulatum essential oil increased, the effect on oviposition reduction increased, but the egg hatching rate had been dropped from 80 to 38% when p< 0.05. Data revealed that as the concentration of the essential oil grew, the rate of larval hatching dropped, which affected the reproductive index and, thus, the rate of reproduction, as illustrated in Table 3.

As illustrated in Figure 4, all concentrations of A. subulatum essential oil showed significant (p< 0.05) results against repellency of Hyalomma ticks except 0.31% concentration. The highest activity was observed at 5% concentrations of A. subulatum essential oil. RC50 and RC90 were also calculated by using probit analysis with regression equation which showed 2.68 and 19.49% values.

The efficacy of a test substance against ticks was measured through the product effectiveness parameter. Different concentrations of A. subulatum essential oil were used and proved to have different results in relation to effectiveness against Hyalomma species. The 5% concentration showed the best results against Hyalomma ticks, which had non-significant (p>0.05) results from the positive control (0.1% cypermethrin) as shown in Figure 5.

Infestation of arthropods, especially arachnids, causes huge economic loss globally (Jabeen et al., 2022; Naseer et al., 2022; Mehnaz et al., 2023). The tick infestations have been controlled by synthetic acaricides over the years, but ticks have developed resistance, which led to the discovery of alternative methods of tick control, especially

Table 3: Effect of various concentrations of A. subulatum essential oil on reproductive parameters and larval hatchability.

IO, Inhibition of oviposition; EH/LH, Egg hatchability/larval hatchability; ILH, Inhibition of larval hatchability; RI, Reproductive index; REI, Reproductive efficiency index; NI, Nutrient index; C1, A. subulatum oil 0.31%; C2, A. subulatum oil 0.625%; C3, A. subulatum oil 1.5%; C4, A. subulatum oil 2.5%; C5, A. subulatum oil 5%; CN, control negative; CP: control positive. Mean±SD along with the same superscripts have a non-significant difference (p>0.05) from each other.

botanical agents, including essential oil (Ibrahium et al., 2022; Akhtar et al., 2023). These essential oils are a volatile mixture of organic compounds that have a mechanism of action and target the ticks in a variety of ways (Faraone et al., 2020; Catani et al., 2022; Özüiçli et al., 2023). A. subulatum essential oil was extracted by hydro-distillation technique and its composition was determined by GC-FID. The major components found in A. subulatum were limonene and α-terpinene. These compositions are like previous studies (Thinh et al., 2021; Bhutia et al., 2022) but variation in the quantity of different components is because of the various factors such as genotype, geographic conditions, harvest time, drying techniques, extraction techniques, and storage conditions (Li et al., 2021; da Silva et al., 2022; Jimayu, 2022). These factors have an impact on the content and composition of the essential oils (Abou Chehade et al., 2022). The other GC-FID detected components along with their quantity in percentage and retention time are listed in Table 1. In the present study, different dilutions of A. subulatum essential oils were prepared in absolute alcohol. The response to adult and larval Hyalomma ticks was dose-dependent (Figure 2 and 3). In the mortality time graph, a significant (P<0.05) difference was observed between higher concentrations (2.5 and 5%) of A. subulatum and the negative control (absolute alcohol) and concluded that higher doses caused greater mortality of adult ticks and larvae. Alruhaili et al. (2023) reported that chemical constituents obtained from A. subulatum were very effective against insects, particularly Tribolium castaneum. In a similar study, Syzygium aromaticum essential oil had shown 100% tick mortality when it was used in 100mg/mL dose (Ferreira et al., 2018). On the other hand, a dose-dependent response was also observed in terms of egg production, egg hatchability, inhibition of larval hatchability, reproductive index, reproductive efficiency, and nutrient index. Nutrient index value was also decreased when the concentration of A. subulatum increased. The acaricidal activity of tested essential oil may be because of the main component’s limonene and α-terpinene which cause the inhibition of acetylcholinesterase enzyme activity in ticks (da Silva Lunguinho et al., 2021) while other compounds such as α- phellandrene, linalool, β-pinene, and 1, 8-cineole have minor inhibitory activity, but they have synergistic action (Wojtunik-Kulesza et al., 2019). The reduction in the egg numbers was because of the female mortality in the first few days after A. subulatum oil treatment. The reduction in oviposition led to a drastic reduction in reproductive index and reproductive efficiency of the Hyalomma ticks when concentrations of essential oil are increased. Essential oils induce cuticular waxes to break and plug the ticks’ respiratory spiracles, which causes water stress and asphyxia (Al-Hoshani et al., 2023b). Additionally, these oils enter the cuticle, diffuse into the haemolymph, and are then transported to internal organs including the ovaries and salivary glands, impairing the digestion and reproductive systems (Jesser et al., 2017).

Like acaricidal activity, a dose-dependent response of A. subulatum against the repellency of Hyalomma ticks was observed for 5 different concentrations. 5% concentration of A. subulatum produced effective response against Hyalomma ticks and showed significant (p<0.05) difference from the DEET treatment as shown in Figure 4. RC50 and RC90 were also calculated by using probit analysis with regression equation which showed 2.68 and 19.49% values. The repellency of A. subulatum is due to the active component limonene and other active components present in essential oil. Terpinene and limonene in the essential oils have proven repellent efficacy against Ixodes ticks (da Silva Lunguinho et al., 2021; Oladipupo, 2022) and these were major components of essential oil of A. subulatum in this study (Table 1). These volatile substances create vapor barriers, driving arthropods away from the essential oil and giving them repelling properties (Salman et al., 2020).

The result of this study indicates that A. subulatum essential oil is a possible approach option for either eliminating or suppressing Hyalomma tick infestation. Further investigation is required to confirm these findings before recommending for commercial application. Moreover, methods to improve the longevity of essential oils as well as processes for improving oil yield following extraction needs to be improved.

All authors are fine with the current version of the manuscript and give their consent for publication.

Not applicable.

The authors have declared no conflict of interest.