Evaluation of Artemisia absinthium L. Essential Oil as a Potential Novel Prophylactic against the Asian Citrus Psyllid Diaphorina citri Kuwayama

Simple Summary

The extensive use of synthetic chemical pesticides to manage insect pests has adversely affected humans and the environment. Therefore, botanical pesticides could be helpful as alternative tools for integrated pest management since they have low mammalian toxicity and minimal risk of developing resistance in target pests. Our current study aimed to evaluate the efficacy of Artemisia absinthium essential oil (AAEO) as a novel alternative to synthetic insecticides against Diaphorina citri. The results indicated that AAEO could be developed as a valuable novel crop protectant against D. citri.

Abstract

Interest in developing novel crop protectants has increased in the recent decade due to the harmful effects of synthetic pesticides on humans and the environment. Diaphorina citri threatens the citrus industry worldwide and is the primary vector of phloem-limited bacterium (HLB). However, there is no available cure for HLB. Diaphorina citri management mainly depends on the use of synthetic insecticides, but their massive application leads to resistance in pest populations. Therefore, alternative pest management strategies are needed. Our results indicated that fewer D. citri adults settled on plants treated with AAEO than on control 48 h after release. The psyllids fed on citrus leaves treated with AAEO significantly reduced the honeydew production compared to the control. The AAEO showed potent ovicidal activity against the D. citri eggs with LC₅₀ 5.88 mg/mL. Furthermore, we also explored the fitness of D. citri on AAEO-treated and untreated Citrus sinensis by using two-sex life table tools. Our results revealed that the intrinsic rate of increase (r) was higher on untreated seedlings (0.10 d⁻¹) than those treated with an LC₂₀ concentration of AAEO (0.07 d⁻¹). Similarly, the net reproductive rate (R₀) was higher for untreated seedlings (14.21 offspring) than those treated (6.405 offspring). Furthermore, the AAEO were safer against Aphis mellifera, with LC₅₀ 35.05 mg/mL, which is relatively higher than the LC₅₀ 24.40 mg/mL values against D. citri. The results indicate that AAEO exhibits toxic and behavioral effects on D. citri, which can be a potential candidate for managing this pest.

1. Introduction

The Diaphorina citri Kuwayama (Hemiptera: Psyllidae) is a well-known significant citrus pest across the globe. Huanglongbing (HLB) is the most devastating disease of Citrus spp. worldwide, limiting commercial production [1]. The HLB-infected trees show abnormal growth, yellowing of leaves, mottling of leaves, and lack of fruit juice. Eventually, the whole plant may die [2,3]. However, no cure for HLB has been reported [4] and controlling D. citri is one of the effective ways to manage HLB [5]. Currently, the primary control measures of D. citri heavily rely on synthetic insecticides [6,7].

Since 2005, the citrus industry in China, Brazil, and Florida has been massively devastated by HLB, which caused a loss of 74% of production [8]. The management of D. citri has played a significant role in HLB dispersal in citrus groves. Eight to twelve insecticide applications are commonly practiced in one cropping season in major citrus-growing countries worldwide [9]. However, the massive and repetitive use of these synthetic insecticides results in environmental pollution and the development of resistance [9,10]. Therefore, there is a need to develop novel eco-friendly alternatives for managing D. citri.

Since ancient times, botanical pesticides have been used to manage various agricultural and household pests, including Azadirachta indica and Nicotiana tabacum [11,12]. Botanical pesticides are safe because they cause no or minimal toxicity to humans and mammals [13]. Furthermore, these pesticides are biodegradable and do not produce any toxic chemicals in the environment [14].

In the recent decade, essential oils (EOs) have been gaining attention among the scientific community as novel alternatives to synthetic chemicals due to their rapid biodegradability and ability to disrupt insect biochemical, physiological, and behavioral functions [15,16]. Artemisia absinthium L. is an aromatic and medicinal herb and it has been reported to have toxicity against various agricultural and household pests, including store pests Callosobruchus maculatus and Bruchus rufimanus [17], repellent and larvicidal activity against Aedes aegypti and Musca domestica [18], contact and residual toxicity against Sitophilus oryzae [19], and fumigant mode of action against Solenopsis geminata and Tribolium castaneum [20].

Therefore, our study aimed to evaluate the A. absinthium (AAEO) toxicity and behavioral effect against D. citri. Furthermore, the effect of AAEO on the fitness of D. citri was also studied using two-sex life table tools.

2. Materials and Methods

2.1. Insects

The D. citri adults were collected from Murraya paniculata L., grown at the South China Agricultural University Guangzhou, China. The psyllids were allowed to acclimatize to laboratory conditions (28 ± 2 °C, 65 ± 5% relative humidity (RH), photoperiod 14:10 (Light: Dark)). Males and females were separated based on their morphological characteristics. The yellow or orange color of the female abdomen indicates that it contains eggs [21,22].

2.2. Plant Materials Extraction Procedure

The aerial parts of A. absinthium were collected from Skardu Baltistan, Pakistan, in August 2022. Our previously published paper reported the plant species and its GC-MS analysis [23] (

Table 1. AAEO dominant constituents were identified through GC/MS [23].

Peak #	Compounds Name b	Relative %	RT a	KI (Exp) c
1	β-myrcene	0.86	12.351	1147
2	Pinocarvone	0.62	13.291	1172
3	α-Gurjunene	1.68	21.838	1416
4	α-Humulene	0.94	22.976	1452
5	α-Copaene	3.51	23.811	1478
6	g-Curcumene	0.45	24.054	1486
7	epi-Cubenol	2.67	24.754	1508
8	β -Calacorene	2.10	26.599	1570
9	(-)-Spathulenol	1.94	26.737	1575
10	Germacrene-D-4-ol	3.48	26.861	1579
11	Guaiol	19.34	27.559	1602
12	Thujol	2.69	27.837	1620
13	4-epi-Cubedol	1.68	28.356	1631
14	Cubenol	1.89	28.64	1641
15	γ-Eudesmol	1.19	29.011	1654
16	8-epi-γ-Eudesmol	1.14	29.113	1657
17	a-Cadinol	2.76	29.269	1663
18	Geranial	8.83	29.844	1686
19	Chamazulene	5.94	31.067	1728
20	1,3-Dicyclopentylcyclopentane	0.93	31.455	1746
21	Fraganol	0.95	32.355	1769
22	Tetrakis(1-methyl)-Pyrazine	2.26	32.92	1797
24	Cubedol	1.16	36.568	1941
25	Geranyl-p-Cymene	1.63	36.748	1948
26	Nerolidol-epoxyacetate	1.12	37.999	1999
27	Geranyl-α-terpinene	5.64	38.176	2007
28	Spathulenol	0.83	39.549	2066
29	Heneicosane	1.60	40.341	2100
30	Eugenol	1.21	40.507	2102
31	Carvacrol	5.47	41.557	2147
32	α-Bisabolol	6.17	41.721	2166
33	1-ethyl-4-methoxy-benzene	0.53	43.784	2256
34	Tricosane	1.48	44.735	2300
35	1-Heptatriacotanol	1.03	44.931	2309
36	Pentacosane	2.20	48.786	2500
37	Heptacosane	1.28	52.539	2700
38	Nonacosane	0.80	56.106	2899
	Total identified	99.9
	Oil yield (%)	0.46
	Monoterpenes	20.42
	Sesquiterpenes	52.69
	Others	26.89

^a Retention time. ^b Compounds are listed in order of their retention time. ^c Retention index relative to C₇-C₄₀ n-alkanes on a DB-1 (30 m × 0.22 mm i.d., 0.25 µm film thickness). Identification methods: RI, based on comparison of calculated RI with those reported in Adams or NIST 08 and previous literature.

). The EOs were stored in transparent glass vials and kept at 4 °C for further experimentation.

2.3. Settling Behaviour of D. citri

The attractiveness and settling behavior of D. citri adults towards sweet orange seedlings (10–15 cm in length) treated with desired concentrations 0.1, 0.5, 1, 2, 3% w/v of AAEO, diluted in 20% ethanol containing 0.01% Tween 80, which corresponds to the dosage of 1, 5, 10, 15, 20 and 30 mg/mL, respectively, were observed in a choice experiment under controlled laboratory conditions (27 ± 2 °C, 65 ± 5% RH, photoperiod 14:10 (Light: Dark). One hundred adults (50 male and 50 female) were released into the center of cages, each cage with six C. sinensis seedlings. Each seedling was sprayed using a mini plastic trigger sprayer (Deqing Yuanchen Plastic Products Co., Ltd., Deqing, China) with 1 mL of the desired concentrations of AAEO and was allowed to dry under the hood. The flasks were randomly positioned inside the cage. There was a total of five replicate cages. The total numbers of D. citri adults settling on each seedling were recorded after an interval of 12 and 24 h after release. Within 2 h after release, the cages were examined to check the mortality due to mechanical injury while aspirating was discarded. The guava crude methanolic extract at 30 mg/mL concentration was used as a positive control, as much literature indicates that Guava repels D. citri [24,25]. The number of D. citri adults that settled on each seedling under the various treatments was compared.

2.4. Antifeedent Activity of AAEO against D. citri

The amount of honeydew excreted by the adults while they were kept to C. sinensis seedlings treated with different concentrations of AAEO was used to assess the feeding activity of D. citri. The feeding bioassay arenas comprised 1.5% agar solution-coated mini glass Petri dishes [26]. Freshly excised leaves from C. sinensis were used for all bioassays. The leaf disk, average size 5.50 ± 0.3 cm in length, was dipped for 5 s in the desired concentrations of AAEO and allowed to dry for one hour under the fume hood. Ten CO₂ anaesthetized D. citri adults were released in each Petri dish, and the Petri dishes were capped with a lid lined with 60 mm filter paper. One hour after release, the Petri dish was examined to check the mortality due to mechanical injury while aspirating was discarded. Then, the Petri dishes were closed with lab parafilm and turned upside down. The filter papers were removed and immersed in 1% w/v ninhydrin 48 h after release for three minutes and were then dried at room temperature [27]. The feeding activities of D. citri were measured by calculating the number of purple spots.

2.5. Ovicidal Toxicity of AAEO

Ovicidal activities AAEO against D. citri were evaluated by confining the eggs containing sprayed C. sinensis seedlings with an aerial insect net (25 cm length, 20 cm width). Sixteen mixed populations (eight male and eight female) of D. citri adults were aspirated. The psyllids were released on the potted seedlings to lay eggs, while the pots and seedlings were covered with an insect net. The psyllids were removed from the gauze nets five days after release, and the number of eggs laid was calculated. The seedlings with eggs were treated with desired concentrations of 0.1, 0.5, 1, 2, and 3% w/v of AAEO. Then, the seedlings were confined with gauze nets and the number of hatched nymphs was counted until all the eggs were hatched. The ovicidal activity was assessed regarding egg mortality rate (EMR) using the formula below.

$$\mathrm{EMR}(\%)=\frac{Number of eggs unhatched }{Total number of eggs laid}\times 100.$$

2.6. Effect of AAEO on Fitness and Development of D. citri

The laboratory conditions were equal to those during the ovicidal toxicity bioassay. Briefly, the C. sinensis seedlings were sprayed with an LC₂₀ concentration (8.3 mg/mL) of AAEO. After drying, D. citri virgin adults (five male and five female) were released in the bioassay arena for mating and oviposition. Five days after release, the adults were removed, and the number of eggs laid in each cage was calculated. The seedlings were observed daily until adult emergence was completed as well as the data of development time from eggs to adult formation, after adult formation as pre-oviposition. The following equations [28,29] were used to compute the population growth rate (PGR), oviposition, and fecundity using the age stage two-sex life table software.

$$PGR=\frac{\left(Nf/N\mathrm{o}\right)}{\mathsf{\Delta }t},$$

whereas

• N_f = Final number of D. citri;
• N₀ = Initial number of D. citri;
• Δt = Total number of days for the experiment.

The result with positive values indicated an increasing population, PGR = 0 indicated a stable population, while negative values indicated a decline in population and led towards extinction.

2.7. Toxicity of AAEO against Non-Targeted Organisms

To evaluate the toxicity of AAEO against Apis mellifera, no-choice feeding bioassays were used [30] under laboratory conditions in a plastic container (0.5 L) by following the procedure described previously [31]. Briefly, the following concentrations of AAEO (24, 36, 48, 60, and 72 mg/mL) were prepared in a 50% sugar solution. Ten healthy foraging workers were introduced into the cage. Each concentration was repeated thrice, and the control contained only 50% sugar solution. The mortality data were recorded within 48 h after treatment. Each container was considered a single treatment and each treatment was replicated five times. Five replications of each container were used to represent one treatment. Imidacloprid was used as a positive control at the following concentrations (5, 10, 15, 20 and 25 ug/mL).

2.8. Statistical Analysis

Chi-square goodness of fit tests were used to estimate the significance of choice between treated and untreated seedlings. The toxicity data were assessed by using Probit analysis (SPPSS 17.0). According to Levene’s test, all data sets were homoscedastic and the mean difference between treatments was separated by using Tukey’s HSD test. The population parameters of D. citri were estimated using the age stage two-sex life table program. The population and age stage parameters of D. citri, e.g., R₀, r, k and s_xj, f_xj, l_x, m_x, e_xj, v_xj, respectively, were calculated as described in the methodology [32]. The two-sex life table was calculated using the following formulae:

$$l_x={{\displaystyle \sum }}_{j=1}^k{S}_{xj}$$

(1)

$$m_x=\frac{{{\displaystyle \sum }}_{j=1}^k{S}_{xj }{f}_{xj}}{{{\displaystyle \sum }}_{j=1}^k{S}_{xj}}$$

(2)

$$R_0={{\displaystyle \sum }}_{x=0}^{\infty }{l}_x{m}_x,$$

(3)

where k is the number of stages. This study used the Euler–Lotka formula’s iterative bisection approach to estimate the r, with the age index starting at 0, as in Equation (2).

$${\displaystyle \sum }_{x=0}^{\infty }{e}^{-r\left(x+1\right)}{l}_x{m}_x=1$$

(4)

$$e_{xj}={{\displaystyle \sum }}_{i=x}^{\infty }{{\displaystyle \sum }}_{y=j}^k{s^{\prime }}_{iy}$$

(5)

$$V_{xj}=\frac{e^{r\left(x=1\right)}}{S_{xj}}{{\displaystyle \sum }}_{i=x}^{\infty }{e}^{-r\left(x+1\right)}{{\displaystyle \sum }}_{y=j}^k{s^{\prime }}_{iy}{f}_{iy}.$$

(6)

3. Results

3.1. Effect of AAEO on Settling Behavior of D. citri

Overall, concentration and time-dependent effects were observed in the settling behavior of D. citri adults. The settling behavior of D. citri adults was not significantly different among the various AAEO concentrations tested compared with the control at 24 h (F = 18.98; df = 4, 24; p = 0.243) after release. However, significant differences were observed after 48 h (F = 66; df = 4, 24; p = 0.005) and 72 h (F = 86; df = 4, 24; p = 0.001) after release (Figure 1). On control plants, compared to those treated with AAEO, more D. citri adults were recorded after 72 h.

3.2. Effect of AAEO on D. citri Feeding Activity

The feeding activity of D. citri measured by the amount of honeydew extraction was presented in (Figure 2). The results indicated a concentration-dependent antifeedant effect of AAEO on the feeding activity of D. citri. Except for 1 mg/mL of AAEO, all the treatments, including 5, 10, 20, and 30 mg/mL, reduced the excretion of honeydew significantly in comparison to the control (F = 84.47; df = 4, 24; p < 0.0001). However, there was a 92 and 86% honeydew excretion reduction by AAEO at 20 and 30 mg/mL.

3.3. Effect of AAEO on Eggs Hatchability of D. citri

The results indicated that a concentration-dependent response of AAEO on the egg hatchability of D. citri was observed. The AAEO has shown potent ovicidal activity with an LC₅₀ 5.88 mg/mL. The number of eggs hatchability per plant was significantly lower than for the control, except for 1 mg/mL AAEO (F = 63.82; df = 5, 29; p < 0.0023). On sweet orange potted plants treated with 30 mg/mL AAEO, only 11.75% of eggs were able to hatch into adults, followed by 5, 10, and 20 mg/mL, where only 30.44, 72.46, and 83.65% of eggs were able to hatch into adults, respectively.

3.4. Population Parameters

The effect of AAEO on the population parameters of D. citri indicated that there was an increase in the intrinsic rate (r), which was higher on untreated sweet oranges (0.10 d⁻¹) than on those treated with an LC₂₀ concentration of AAEO (0.07 d⁻¹). Similarly, the net reproductive rate (R₀) was higher for untreated C. sinensis seedlings (14.21 offspring) than for those treated (6.40 offspring) with LC₂₀ concentration of AAEO (

Table 2. Effect of AAEO on reproductive and population parameters of the D. citri on treated sweet orange seedlings compared to untreated sweet orange seedlings.

Traits	Treated	Untreated
r (per day)	0.07	0.10
ʎ (per day)	1.07	1.11
GRR (offspring)	7.53	16.02
R0 (offspring/individual)	6.40	14.21

r; The intrinsic rate of increase (per day) ʎ; The finite rate of increase (per day) GRR; Gross reproductive rate (offspring) R₀; The net reproductive rate (offspring/individual).

).

Diaphorina citri, the comprehensive age-stage survival rate (sxj) on treated and untreated C. sinensis seedlings, was determined. Our findings showed the possibility of a freshly hatched larva making it to age x and stage j (Figure 3) because development rates varied across individuals on treated and untreated seedlings; similarly, the projected curves exhibited completely different layouts at each developmental stage. Individual survival rates rapidly dropped with age and showed an inverse relationship between treated and untreated seedlings (Figure 3). The developmental time of D. citri females was long, and the survival rate was shorter on the untreated than the treated ones, while in the case of males, the development was shorter, and the survival rate was longer on untreated compared to on treated C. sinensis seedlings (Figure 3).

The highest value of age-stage specific fecundity (f_xj) was higher on untreated sweet oranges than on treated ones (Figure 3). There is a direct relation to the age-specific maternity (l_x*m_x) of D. citri. As the survival rate increases, fecundity increases in treated and untreated cases. However, the constant peak point of age-specific maternity (l_x*m_x) of D. citri was higher on untreated sweet oranges than on C. sinensis seedlings treated with AAEO (Figure 4).

The effects of treated and untreated sweet oranges on the population’s expected average life expectancy (e_xj) at egg, nymph, and adult stages of D. citri were determined in (Figure 5). The longevity of the newly hatched D. citri eggs was longer in untreated sweet oranges than in treated ones. The peak life expectancy (e_xj) value for female adults of D. citri was higher on untreated sweet oranges than on treated oranges (Figure 5). The exj of male adults of D. citri was the maximum on treated sweet oranges compared to untreated oranges. Overall, all stages of the highest life expectancy (e_xj) of D. citri were recorded on untreated sweet oranges (Figure 5). The age-stage reproductive value (v_xj) of D. citri in (Figure 6) explains an individual’s role in the future population (i.e., the population forecasting scale) at age x and stage j.

3.5. Toxicity of AAEO against A. mellifera

The AAEO caused toxicity against A. mellifera at significantly higher concentrations, with LC₅₀ and LC₉₀ of 35.05 and 55.86 mg/mL, respectively, which is too high compared to the lethal dose of AAEO against D. citri LD₅₀ of 5.20 µg/insect via the topical application method recorded in our previous published paper [33] (

Table 3. Toxicity of AAEO against A. mellifera.

Concentration (mg/mL)	Exposed a	% Mortality ± SD b	LC50 c (95% CL d)	LC90 e (95% CL)	X² (df) f	p-Value
24	64	21.65 ± 0.87	35.05 (25.58–34.69)	55.86 (46.01–67.81)	0.94 (2)	0.23
36	60	35.32 ± 0.59
48	67	52.01 ± 0.87
60	67	65.34 ± 0.60
72	63	84.32 ± 0.51
Control	65	4.11 ± 0.11
Imidacloprid (ug/mL)
5	66	20.22 ± 0.23	13.49 (9.45–19.34)	47.87 (33.32–55.31)	4.41 (3)	0.49
10	58	29.11 ± 0.35
15	69	48.04 ± 0.45
20	61	65.21 ± 0.32
25	56	81.00 ± 0.13
Control	62	7.12 ± 0.99

^a Total number of bees treated. ^b SD. Standard deviation. ^c LC₅₀. 50 % lethal concentration. ^d CL. Confidence limits. ^e LC₉₀. 90% lethal concentration. ^f χ² chi-square, df degrees of freedom.

). Therefore, AAEO can be considered safe against A. mellifera.

4. Discussion

Botanical pesticides are plant-based substances, including pyrethrin, azadirachtin, neem, garlic, and vegetable oil. Because they degrade quickly in the presence of light and air, botanicals often have a short shelf life in the environment. [34]. These include plant extracts and essential oils, which are eco-friendly, biodegradable, and nontoxic to mammals [33]. The essential oils from various plants are reported to have antifeedant, repellent, and toxic activities against many insect pests [35]. Eos are complex mixtures of different compounds but are majorly dominated by monoterpenes and sesquiterpenes [36]. These monoterpenes and sesquiterpenes exert different toxic and behavioral effects against insects. For example, limonene decreased oviposition in mite Oligonychus ununguis [37], carvacrol and thymol showed contact toxicity against Pochazia shantungensis [38], α-pinene, eucalyptol and camphor exerted both fumigant and antifeedant activities against Solenopsis invicta and Meloidogyne incognita [39], thymol showed substantial contact toxicity against Blattella germanica (Yeom et al., 2012), and 1,8-cineole showed a fumigant mode of action against Solenopsis invicta and Ectomyelois ceratoniae [40,41]. Similarly, carvacrol showed contact toxicity against D. citri [33].

Plant volatiles are crucial in herbivore host location and recognition [42]. Odors and plant colors mediate how herbivore insects find and recognize their potential host [43]. The D. citri relies on its olfaction to locate and evaluate its potential host [44]. The volatile chemicals released by non-host plants obscure the host plant odor that phytophagous insects detect, leading to host plant avoidance and non-preference [45]. Regarding the repellent activity of AAEO against D. citri, results indicated a concentration-dependent effect. The adults strongly preferred settling on the control C. sinensis seedlings to the treated seedlings. Compared to the control, the adult D. citri settling did not significantly decrease 24 h after release. However, only a few adults were seen on the treated plant 48 and 72 h after release compared to the control because it took D. citri just around 9 h to distinguish volatiles from host plants, from a combination of volatiles from non-hosts in the open atmosphere [46]. A literature report indicated that the host finding and recognition ability of D. citri were reduced when non-host plant semiochemicals were used [45,47]. Many non-host plants have shown repellent activities against D. citri, including Guava [3,24,48], Allium spp [49,50]. HLB bacteria can only multiply in the body of the eukaryotic host [51]. The transmission of HLB bacterium from the infected to the uninfected tree was primarily taken by nymphs and adults of D. citri [52]. Here, we found that AAEO reduces the feeding activity of D. citri, measured as the number of purple spots on the treated leaf disc. AAEO at 20 and 30 mg/mL reduced honeydew secretion by 72.86 and 85.5%, respectively. However, the effect of AAEO on D. citri feeding in terms of the number of honeydew droplets recorded per filter paper disc was lower than cyantraniliprole, a synthetic anthranilic diamide insecticide, which caused an 80% reduction in honeydew droplets secretion by D. citri at 0.1 µg/mL [53]. To better understand the antifeedant activity of AAEO against D. citri, further investigation should be conducted using electrical penetration graph (EPG) technology.

Essential oils (EOs) are effective against several insect species. They act as growth inhibitors, toxins, deterrents, repellents, and toxicants [54]. The EOs of azadirachtin and Piper aduncum against nymph and adults of D. citri caused 90–100% mortality in nymph and below 80% in adults, being nontoxic to ectoparasitoid Tamarixia radiata (Hymenoptera: Eulophidae) [26]. Similarly, Syzygium aromaticum, Eucalyptus obliqua, Tithonia diversifolia, and Citrus limoniaI EOs showed considerable toxicity and repellent effects against D. citri [55,56]. Primarily D. citri management was prodigiously focused on controlling adult psyllids. For this, many classes of insecticides have been utilized [57]. Limited literature is available regarding the developmental and ovicidal products against D. citri. According to the current study, AAEO-treated potted C. sinensis seedlings exhibit concentration-dependent ovicidal action. When enclosed with the dry residue of AAEO at 20 and 30 mg/mL, respectively, only 11.75 and 30.44 eggs were able to hatch into adults, whereas in the control and 10 mg/mL concentrations, the hatching percentages were 93.45 and 93.78, respectively. The result showed that AAEO has ovicidal activity against D. citri. However, AAEO ovicidal activity was lower than that of cyantraniliprole, a synthetic anthranilic diamide insecticide, which caused complete inhibition of D. citri eggs’ hatchability at 0.025 µg/mL [53].

The potential mechanism of action of EOs against insects is neurotoxicity [58,59]. These EOs typically contain complex combinations of sesquiterpenes, biogenetically related phenols, and monoterpenes. These compounds have a variety of hydrophilic and hydrophobic properties that can easily permeate insect cuticles and disrupt their physiological processes [60,61]. Despite the most promising properties of EOs as a natural insecticide, many technical issues are raised for their broader application due to their rapid volatility and poor water solubility [15]. There are many challenges and constraints related to the commercialization of EOs, including strict legislation and lack of raw material availability [60]. Their rapid degradability and low persistence may significantly reduce toxicity [60], transient effects, and a dearth of high-quality raw materials that are affordably priced [61]. Due to their rapid breakdown and short persistence, EOs may have significantly lower toxicity [61]. However, the efficacy and persistence of EOs can be enhanced by encapsulation, nanoparticles, and nano gel formulation, and cyclodextrins [62]. Overall, EOs have the potential to develop eco-friendly candidates for novel pest management, which should be a top priority for preserving ecosystems from contamination. EOs and plant extracts are safer for the environment, humans, and non-targeted organisms than synthetic insecticides [63]. The AAEO caused toxicity against A. mellifera at significantly higher concentrations, with an LC₅₀ value of 35.05 mg/mL, which is too high compared to the LD50 value of 5.20 µg/insect of AAEO against D. citri.

5. Conclusions

The current study found that AAEO had insignificant toxicity toward honeybees when evaluated as non-targeted organisms and demonstrated repellent and ovicidal properties against adult D. citri. More research should be pursued regarding its broader applicability and effect on natural enemies. It was concluded that the AAEO might be developed as a novel prophylactic against D. citri with the edge of being environmentally friendly.