Novel acaricidal and growth-regulating activity of Aloe vera and Rheum rhabarbarum extracts and their oil/water nanoemulsions against the camel tick, Hyalomma dromedarii

Hyalomma dromedarii is an important tick species infesting livestock. This work evaluated the novel adulticidal, insect growth-regulating, and enzymatic efficacy of ethanol plant extracts of Aloe vera and Rheum rhabarbarum and their nanoemulsions against males and engorged females of the camel tick, H. dromedarii. The physicochemical properties of nanoemulsions were evaluated. The High-Performance Liquid Chromatography (HPLC) analyses indicated that the extracts contained polyphenols and flavonoids, which could enhance their acaricidal effect. Dynamic light scattering (DLS) of the nanoemulsions of A. vera and R. rhabarbarum were 196.7 and 291 nm, whereas their zeta potentials were − 29.1 and − 53.1 mV, respectively. Transmission electron microscope (TEM) indicated that nanoemulsions showed a regular spherical shape (less than 100 nm). Fifteen days post-treatment (PT) with 25%, the mortality% of A. vera and R. rhabarbarum were 88.5 and 96.2%, respectively. Five days PT, the median lethal concentration values of A. vera, R. rhabarbarum, and their nanoemulsions were 7.8, 7.1, 2.8, and 1.02%, respectively, and their toxicity indices were 91.02, 100, 36.4, and 100%, respectively. Their median lethal time values PT with 3.5% were 6.09, 5.09, 1.75, and 1.34 days, respectively. Nanoemulsions enhanced the efficacy of the crude extract 1–7 folds, 5 days PT, and accelerated their speed of killing ticks 2–4 times. The total protein and carbohydrates, Acetylcholinesterase, Alpha esterase, and Amylase were affected PT. The reproductive potential of engorged females was adversely impacted. In conclusion, the novel A. vera and R. rhabarbarum extracts were promising acaricides, and their nanoformulations enhanced their efficacies.


Chemical and biochemical analysis
Chemical Oleic acid 90%, polysorbate 20 (Tween 20), Sodium Glycocholate 97.5%, Sodium Cholate 99%, and Distilled water (de-ionized), all chemicals were purchased from Alfa Aesar, Karlsruhe, Germany, and used with no further purification.Chemicals used for biochemical analysis were the Bovine albumin standard, purchased from Stanbio Laboratory (Texas, USA); Commasie brilliant blue G-250, purchased from Sigma (Sigma Chemical Co.); P-nitroanisole (purity 97%), acquired from Ubichem Ltd. (Ham pshire); and nicotinamide ademine dinucleotide phosphate (reduced form, NADPH), got from BDH chemicals Ltd. (Poole, England).The rest of the chemicals were of high quality purchased from commercial local companies and used without further purification.

Synthesis of plant extracts
Synthesis of plant extracts before nanoformulations.Both A. vera leaves and R. rhabarbarum stems were separately washed with distilled water twice and attained to be dehydrated.After complete dryness for three days at 50 °C in a vacuum oven, the plants were ground to a fine powder and washed several times with distilled water.About 250 g of each plant was placed in a beaker containing about 600 ml of 10% ethanol (v/v).The beaker was transferred to a hotplate and the temperature was raised to 50 °C for 3 h with occasional mixing using a glass rod (every 10 min) with flipping the flakes up and down to achieve good extraction.Afterward, the beaker was attained to cool to room temperature and then cooled at a temperature of 5-10 °C for two hours.The cooled beaker was filtered several times using a cotton tissue and then filtered using a Whatman filter paper; the supernatant was re-concentrated using a vacuum rotary evaporator to 50 ml.The net solution was kept in a dark glass bottle and kept at a temperature of 5-10 °C.For easier concentration manipulation and definite solid-content weight quantification, a small volume of 15 ml (pre-weighted) was re-concentrated using freeze drier utile complete solvent evaporation and collection of solid contents.
Preparation of extract-nanoemulsions.Preparation of nanoemulsions of the plant extracts was done according to a previous portocol 65 with little modifications, as follows: 2 gm of oleic acid, placed in a 50 ml beaker, was heated to 40 °C (solution I).On another 50-ml beaker, 2.5 ml solution of Tween 20 and 15 ml of concentrated extract were added to a well-stirred mixture consisting of 0.7 gm sodium glycolate, and 0.7 gm sodium taurocholate dissolved in 10 ml water, a portion-wise addition, and then the overall mixture heated to the same temperature 40 °C (solution 2).Solution I is then poured into solution II at the same temperature to get a solution of a clear nanoemulsion at 40 °C, which in turn is dispersed using an ultrasonic probe sonicator for 20 min at 500 W with the addition of 100 ml ice cold water.Mannitol was added to the dispersion as a cry protectant and then lyophilization was done to get a semi-solid substance.

Characterization of nanoemulsion
Droplet size and Zeta Potential.The hydrodynamic radius of the synthesized extract nanoemulsions and polydispersity index (PDI) were done by dynamic light scattering (DLS) at a fixed angle of 173° at room temperature.Zeta potential or the surface charge was measured by the frequency shift of scattered light at a scattering angle of 12°.Moreover, PDI, Radius, and Zeta potential were investigated by a Zetasizer nano Zs analyzer (Malvern instruments) at the Egyptian Petroleum Research Institute (EPRI), Cairo, Egypt.About 5-10 mg of each powder was dispersed in 10 mL of distilled water at a temperature of 25 °C.
NLC surface morphology by transmission electron microscope (TEM).The morphology and internal structure visualization of obtained nanoemulsions were investigated using field transmission electron microscopy (HR-TEM, JSM-7100F) at EPRI.Images were recorded with JEOL JEM-2100-115 a high-resolution transmission electron microscope with an accelerating voltage of 200 kV.Nearly 1 µL of NLCs was diluted with double distilled water (1:200) and placed on a 200 mesh carbon-coated grid and attained for two min.and the excess liquid was disposed of by filter paper.One to two drops of 2% (w/w) phosphotungstic acid (PTA) were added to the grid for 10 s to achieve negative staining, the excess PTA was removed via adsorption on a filter paper.

Tick
Adult males and engorged females of H. dromedarii were collected from places around infested camels in Toukh (35 km north Cairo: 30° 21′ 11.6″ N and 31° 11′ 31.5″E), Qalyubia Governorate, Egypt.Ticks were transferred to the laboratory in plastic cups covered by a piece of cotton net gauze.Morphological identification was performed 66 .
N.B.This study involved the treatment of ticks and did not involve live vertebrates.All experiments were accomplished in agreement with the relevant guidelines and regulations of the Ethical Committee of the Faculty of Veterinary Medicine, Benha University, Egypt (BUFVTM 02-10-22 www.nature.com/scientificreports/

Adult immersion tests
Adulticidal effect An in-vitro adult immersion test (AIT) was used to evaluate the toxicity of plant extracts against H. dromedarii in line with a previously described protocol 67 .Five and six concentrations were diluted in distilled water for each plant extract and its nanoemulsions, respectively.Ten active males were immersed for 60 s in a 100 ml solution at each concentration.Three replicates were used for each concentration (30 ticks/ concentration) and the control group was treated with distilled water.After immersion, ticks were added to a Petri dish with filter paper (Whatman no. 1) and kept at 27 ± 2 °C and 80 ± 5% relative humidity.Tick mortalities were checked up to 15 days post-treatment (PT) and recorded as dead when no reaction was shown after stimulation with a fine brush.

Insect growth regulating effect
Tests were carried out to determine the efficacy of plant extracts before and after nanoformulations against engorged females of H. dromedarii, according to a previously described protocol 18 with a slight modification.Five concentrations were freshly prepared in distilled water.Thirty ticks were individually weighted and treated as mentioned in the previously mentioned AIT protocol for each concentration.Each immersed tick was kept uprightly in a labeled vertical test tube covered with a cotton plug at 27 ± 2 °C and 80 ± 5% relative humidity.The weight of the egg mass was measured and the number of hatched eggs was counted.

Biochemical and enzyme assay analyses
Apparatus Ticks were treated with the LC 50 of each tested material and homogenized for biochemical analysis in a chilled glass Teflon tissue homogenizer (ST-2 Mechanic-Preczyina, Poland).After homogenization, supernatants were kept in a deep freezer at -20 °C till used for biochemical assays.A double-beam ultraviolet/ visible spectrophotometer (Spectronic 1201, Milton Roy Co., USA) was used to determine the optical density of the colored substances or metabolic compounds.

Preparation of ticks for analyses
Treated ticks were homogenized in distilled water (50 mg /1 ml); homogenates were centrifuged at 8000 r.p.m. for 15 min at 2 °C in a refrigerated centrifuge.After that, the deposits were discarded and the supernatant (enzyme extract) was stored at < 0 °C for less than a week until used.All experiments contained three replicates (tick homogenates) and the results of biochemical determinations were pooled in triplicates 68 .
Total proteins 69 and total carbohydrates were assessed in an acid extract of the sample by the phenol-sulphuric acid reaction 70 , extracted and prepared for the assays 71 .Acetylcholinesterase (AchE) activity was measured using acetylcholine bromide (AchBr) as a substrate 72 ; Alpha esterases (α-esterases) were determined using α-naphthyl acetate as a substrat 73 .Determination of amylase activity was also revealed 68 .

Data analyses
The data were analyzed through SPSS V23 (IBM, USA) to perform the one-way analysis of variance (ANOVA) (Post Hoc/Tukey's HSD (honestly significant difference) to compare the significant difference within and between groups and the Probit analyses to calculate the lethal concentration (LC) and time (LT) values.All significant levels were set at P<0.05.
The mortality data were corrected 74 according to the following equation: Corrected Mortality% = (MT% − MC%)/ (100 − MC%) X 100 MT: mortality of the treated group; MC: mortality of the control group.The relative toxicities 46 and the toxicity indices were determined 75 for a comparison of the tested extracts, where the most toxic plant extract had given 100 units on the toxicity index scale.

Ethical approval
The protocol of this work was approved by the Ethical Committee related to the Faculty of Veterinary Medicine at Benha University, Egypt (BUFVTM 02-10-22).

Results and discussion
Hyalomma ticks have economic importance 76 .Previous studies indicated that H. dromedarii had acquired resistance against the commercially and widely used acaricides, Deltamethrin (Butox®) 10 and Phoxim® (50%, an analogous dimethyl ester, C 12 H 15 N 2 O 3 PS) in Egypt 11 , in Qalyubia Governorate, Egypt, the same locality of the present study.Consequently, searching for eco-friendly acaricides is a pressing need 12,16,67 .Besides their pesticidal effects, botanicals have fungicidal, bactericidal, and antioxidant properties; therefore, they are used in medicine and cosmetics 15,24,32,49 .There are few studies on the efficacy of herbal extracts against H. dromedarii 10,78-81 and very rare studies tested their effect on its reproductive potential 50 .This study evaluated the innovative acaricidal

HPLC of the ethanol extracts
To identify the components presented in the ethanol extracts of A. vera and R. rhabarbarum extracts in this study, HPLC analysis was carried out with 19 standard polyphenols (Table 1).Active ingredient polyphenols were revealed for A. vera (Table 2) and R. rhabarbarum (Table 3) and the HPLC analysis indicated that both ethanol extracts were enriched with polyphenolic and flavonoid active ingredients that may give a good interpretation of their acaricidal and IGR effects.Alike findings were reported 28,35,81 .The most active ingredients identified by  www.nature.com/scientificreports/GC-MS analysis in A. vera gel extract were Terpene and Sesquiterpene hydrocarbons 82 .Moreover, a combination of active ingredients in the extract could synergistically increase the biological activity of the extract 19,20,51 .This study revealed that A. vera extract was rich in many active ingredients, such as catechin, gallic acid, naringenin ellagic acid, and ferulic acid (567.51, 441.29, 346.56, 273.18, and 210.02 µg/g, respectively) and good amounts of methyl gallate, chlorogenic acid, coumaric acid, syringic acid, daidzein, hesperetin, querectin, apigenin, pyro catechol, rutin, and cinnamic acid (93.59, 90.73, 78.17, 58.09, 58.00, 47.23, 36.88,19.53, 15.69,  13.64 and 3.11 µg/g, respectively).In contrast, kaempferol, vanillin, and coffeic acid had no participation from A. vera extract (Table 2).
Phytochemicals in Rhubarbs in another study included stilbenes, anthraquinones, and flavonoids 59 .The phytochemical characterization of the extracts of Aloe arborescens enabled the identification of the presence of condensed and water-soluble tannins, besides anthraquinones, including aloeresin, aloenin, aloin A and B, homonataloin, and 4′-O-glucosylisoaloeresin. Water-soluble tannins were the main components of the extracts with acaricidal activity 84 .The presence of the phenolic contents may play an important role in the elimination of ticks and mites through their action on the GABA receptor and the octopamine receptor 85 .
Against H. dromedarii in Egypt, Saussurea costus extract contains mainly sesquiterpene, fatty acid esters, phenols, and acyclic hydrocarbons which might explain its pesticidal effect 86 .A similar study showed that myrrh is slightly more effective than ginger against H. dromedarii and this may be due to its high contents of flavonoids and phenols 10 .The total phenolic and flavonoid contents of nine aqueous plant extracts explain their acaricidal efficacy; Ricinus communis possessed the highest total phenolic contents (95.50 ± 0.17 mg/g), while Quercus cortex contained the highest flavonoid contents (70.78 ± 0.17 mg QE/g) 77 .

Physicochemical properties of nanoemulsion-plant extracts
Dynamic light scattering (DLS) and Zeta potential (z.p).According to the "Brownian motion", when a small suspended particle moves through a liquid solution, it will undergo randomized motion because of the collisions by the molecules themself and; consequently, particles.Furthermore, DLS is a very reliable and powerful technique to study the diffusion attitude of the small to relatively large particles in a solution depending on the particle's size (hydrodynamic radii) and shape variations.After size and shape data collection, the data were analyzed internally by the device to obtain the degree of the heterogeneity, or what so-called poly dispersity index (PDI), of the solution based on size variation, which may occur as a result of agglomeration or aggregation.The International Organization for Standardization (ISOs) declared that if the values of PDI were less than 0.05, the solution under investigation is more likely to be monodispersed or homogenous, while values > 0.7 indicated that the solution is commonly of broad size or polydisperse particle distribution 27,87 .In this study, DLS of the prepared A. vera extract nanoemulsion pretended particle size of 196.7 nm with pdi of 0.300, confirmed that the synthesized A. vera extract nanoemulsion had a good particle size with polydisperse particle distribution.Like A. vera extract nanoemulsion, R. rhabarbarum nanoemulsions showed a slightly larger average size of 291nm with little reduction in the pdi value of 0.239, but it was still fit to be described by" polydispersed" particle distribution (Table 4).The stability of the colloidal system related to the dispersion of solid materials in the liquid was monitored with zeta potential.It is caused by the net electrical charges in a specific region in a slipping plane depending on the location of such plane.Zeta potential magnitude indicates the degree of electrostatic repulsion forces between the same adjacent charged particles in the dispersion.For small particles like molecules, a higher zeta potential will promote stability, i.e., dispersion will resist aggregation, and vice versa when the zeta potential is small enough to make the attractive forces exceed the repulsion forces, i.e., particles become closer to each other and the dispersion will be flocculated 34 .Consequently, colloids with high values of zeta potential, even negative or positive, are electrically stabilized while colloids with low zeta potential values have a large tendency to flocculate or coagulate.It is interesting that the results of this study showed values of zeta potential of R. rhabarbarum extract nanoemulsion of −53.1 mV.The larger negatively charged numerical value reflects the higher repulsion forces exerted on the system due to the similar negative charges.Increasing the number and concentrations of polyphenols and flavonoids in the extract promoted extra negative charges because of a wide spread of hydroxyl groups.From this point of view, A. vera nanoemulsion extract contained less concentration of polyphenolic active ingredients that may affect the net charge or zeta potential to be −29.1 mV.Comparatively, the zeta potential of the nanoemulsion of R. rhabarbarum is much greater than that of A. vera indicating that R. rhabarbarum nanoemulsin had excellent stability.In the meantime, both nanoemulsions have good stability attitudes 88 .
Transmission electron microscope (TEM) findings of plant extract-loaded nanoemulsions.The transmission electron microscope is one of the most important analyses to confirm the internal structure of nanoemulsions prepared in this study (Figs. 1 and 2).Nanoemulsions of R. rhabarbarum and A. vera extracts showed regular and spherical shapes in various sizes (less than 100nm); whereas that of R. rhabarbarum presented a slightly higher size near 200 to 600nm confirming the polydispersity depicted by PDI findings.The revealed size in this study came along with data obtained by zeta sizer; the results of DLS measure the average size, not definite particle size like that of TEM 27 .

Acaricidal activity of the plant extracts against H. dromedarii
The mortality data of the present study showed a time and concentration-dependent relationship; similar studies were recorded 12,67 .Three days post-treatment (PT) with 25% of A. vera and R. rhabarbarum, the mortality percentage (MO%) reached 50 and 53.6%, respectively.Five days PT, MO% reached 61.5 and 65.4%, respectively.Meanwhile, 15 days PT, MO% reached 88.5% and 96.2%, respectively (Table 5).Three days PT, the LC 50 and LC 95 values of A. vera were 9.8 and 25.6%; whereas those of R. rhabarbarum were 8.9 and 24.8%, respectively (Table 6).Such values five days PT were 7.8 and 22.9% for A. vera and 7.1 and 21.4% for R. rhabarbarum, respectively (Table 7).The toxicity index values were 90.8% and 100%, at three days PT, and reached 91.02 and 100%, respectively, at five days PT (Tables 6 and 7).Meanwhile, LT 50 values PT with 25% and 3.5% of A. vera were 1.86 and 6.09 days; while those of R. rhabarbarum were 1.65 and 5.09 days, respectively (Table 8).Parallel to our findings, the hydroethanolic extract of the leaves of Aloe rupestris induced an acricidal effect (66.6%) against the cattle tick, Rhipicephalus turanicus (Acari: Ixodidae) at a concentration of 20% (200 mg/mL) 89 .In addition, aqueous extracts (5%) of A. vera, garlic, Allium sativum, and ginger, Zingiber, officinale, against the brown dog tick, Rhipicephalus sanguineus, showed acaricidal effect (100%) against females, males, and larvae and showed cuticular damage along with breaching and loss of homogeneity of both epicuticle and endocuticle 90 .The findings of A. vera in this study came along with some other studies as it has a great potential for development as a botanical acaricide against the carmine spider mite, Tetranychus cinnabarinus (LC 50 values of the acetone  Very recent and similar studies revealed the efficacy of other botanicals as acaricides.Complete mortalities of H. dromedarii and Rhipicephalus (Boophilus) annulatus were recorded PT with 25 mg/mL of Araucaria heterophylla and Commiphora molmol extracts for seven days.The LC 50 values PT of H. dromedarii with the methanol and hexane extracts were 1.13 and 1.04 mg/mL and 1.47 and 1.38 mg/mL, respectively; whereas such values against R. annulatus were 1.09 and 1.41 as well as 1.55 and 1.08 mg/mL, respectively 28 .The influence of the ethanol extracts (25%) of costus, Vitex castus, and, Z. officinale, against H. dromedarii demonstrated that MO% 15 days PT reached 80.8 and 84.7%, respectively; their LC 50 values, five days PT, were 10.50 and 9.60%, respectively; and their toxicity indices reached 91.43 and 100.00%, respectively.On the other hand, their LT 50 values PT with 25% were 2.6 and 2.5 days, respectively 78 .Moreover, the adulticidal effect of olive, Olea europaea L, oil against H. dormidarii males reached 83.33%, 15 days PT with 25%, and its LT 50 and LT 95 were 5.161 and 17.072 days, respectively, whereas its LC 50 and LC 95 values were 12.715 and 46.386%, respectively, 12 days PT 50 .
Analogs to our findings, another species of R. palmatum extract and five of its isolated anthraquinones possessed insecticidal activity against the brown planthopper, Nilarparvata lugens, and the northern armyworm, Mythimna separate.A high amount of R. palmatum compounds (82.87 mg/g) were detected in its acetone extract inducing stronger insecticidal effect than those of the aqueous and ethanol extracts (yields = 8.84, 37.17, and 36.92%,respectively).Amongst the isolated compounds in such study, emodin displayed very high and moderate insecticidal activity, as its LC 50 values were 84.30 and 548.74 μg/mL, respectively).A similar pattern was recorded for toosendanin (LC 50 = 89.34 and 418.24 μg/mL, respectively 93 .In contrast to our superior acaricidal effect, fractions of R. palmatum root extract and anthraquinone (aloe-emodin, emodin, chrysophanol, and physcion) showed no acaricidal activities against the mite, T. urticae 94 .Different outcomes might be related to using different pest species and extracts.Moreover, R. palmatum has fungicidal 93,94 and herbicidal activitie 94 .
A comparable study revealed the acaricidal activity of Citrus limetta seed oil against the cattle tick, Rhipicephalus microplus (LC 50 = 2.87 and 3.96% PT of larvae and adults, respectively) and 100% MO were reached PT with 12.5% 95 .Furthermore, the crude extract along with water and petroleum ether fractions of Areca (A.) catechu seeds were effective against cypermethrin-resistant R. (R.) microplus 53 .In addition, Saussurea costus as methanol and hexane extracts of effectively controlled cattle and camel ectoparasites; MO% of H. dromedarii, seven days, PT with 12.5 and 25 mg/ml was 100 and 90% (LC 50 = 1.37 and 2.33 mg/ml, respectively).In the meantime, such values against R. (B) annulatus were 100 and 93.33% coupled with 1.23 and 1.95 mg/ml, respectively 86 .A comparable acaricidal effect of an aqueous neem extract against Sarcoptes scabiei var.cuniculi in vitro and experimentally-infested rabbits was recorded 14 .www.nature.com/scientificreports/

Insect growth regulating effects of plant extracts against H. dromedarii
After treatment with A. vera and R. rhabarbarum in this study, the reproductive potential of engorged females was adversely affected when compared to that of the control group.After treatment with the highest concentration, 25%, egg production had ceased and engorged female weight reached 54.00 and 51.83 g, respectively (Table 9).Analogs result was recorded as the ethanol extract of A. vera effectively reduced egg production/ female of T. urticae, followed by acetone, methanol, and petroleum ether extracts by 96.0, 94.0, 85.0, and 83.0%, respectively (LC 50 = 0.950, 1.406, 2.115, and 3.312%, respectively).Moreover, ethanol extract was the most effective repellent against T. urticae females 92 .
A similar finding revealed a strong effect on the reproductive parameters represented by a marked decrease in the number of laid eggs PT of fresh and dry Aloe arborescens Mill.extracts (solvents pure ethanol, ethanoldichloromethane binary mixture, and ethanol-dichloromethane-acetone ternary mixture, contained watersoluble tannins) against engorged females of Rhipicephalus (Boophilus) microplus 84 .A related finding revealed that Citrus limetta seed oil significantly (p < 0.001) lowered the oviposition rate, egg hatching, and reproduction efficiency of treated ticks of R. microplus 94 .Furthermore, Azadirachtin, a tetranortriterpene extracted from neem (LC50 = 0.47 ppm) adversely affected the fecundity and the development of the follicular epithelial cells of ovaries of female N. lugens, and the weight of the treated females was significantly reduced, 23, 40, and 64% PT with 0.1, 0.25, and 0.5 ppm, respectively 96 .

Acaricidal activity of nanoemulsions against H. dromedarii
This study indicated the efficacy of nanoemulsions of A. vera and R. rhabarbarum against H. dromedarii PT with 15%; MO% reached 83.3 and 76.7%, respectively, three days PT; 86.6 and 90%, respectively, five days PT; and 100% for both extracts, 12 and nine days PT, respectively (Table 10).The LC 50 and LC 95 values were 4.2 and 17.67% PT for three days with nanoemulsions of A. vera and reached 3.5 and 17.4% PT with nanoemulsions of R. rhabarbarum, respectively (Table 11).Five days PT, the LC 50 and LC 95 values were 2.8 and 15.8% as well as 1.02 and 32.72%, respectively (Table 12).Regarding the LC 50 values, the prepared nanoformulations enhanced the efficacy of the ethanol extracts of A. vera and R. rhabarbarum 2.3 and 2.5 times, three days PT (Table 11), and 2.8 and 7 times, five days PT (Table 12).R. rhabarbarum induced a superior effect and its toxicity index reached 100% (Tables 11 and 12).The LT 50 values of A. vera and R. rhabarbarum were 0.898 and 0.839 days, respectively, PT with 15% and 1.75 and 1.34 days, respectively, PT with 3.5%.Nanoemulsions accelerated the speed of killing of ticks 2-4 times faster than the ethanol extracts (Table 13).
The finding of this study came along with a previous study that used different concentrations of methanol leaf extract, green synthesized silver (AgNPs), and chitosan nanoparticles (CsNPs) using A. vera and Nerium oleander against Musca domestica and indicated that the nanoparticles were more potent than the methanol extract 97 .Furthermore, a corresponding study indicated that the larvicidal effect of silver nanoparticles AgNPs  10 .In addition, AgNPs of R. rhabarbarum have antibacterial activity towards Gram-positive (+ve) strains of Staphylococcus aureus and Gram-negative (−ve) strains of Escherichia coli 98 .14).A similar study proved the IGR effect of AgNPs and CsNPs encapsulated A. vera gel extract after treatment of larvae of M. domestica as they prolonged larval duration and reduced the pupation and adult emergence rates 82 .

Biochemical characterization
Enzymes are usually used as reliable indicators for evaluating the impact of the applied toxic materials against insects 99,100 ; the mechanisms of insecticide resistance mostly include enzymes involved in the detoxification of carbamates, organophosphates, pyrethroids, and growth regulators such as non-specific esterase, Glutathione-S-transferase (GSTs), and P450-mediated monooxygenase (MFOs) 100 .Generally, biochemical analyses of the present work revealed that the total protein, carbohydrates, AChE, Alpha esterase, and Amylase were affected after treatments; their values PT with nanoformulations were significantly (p < 0.05) more affected than those of the control group, except for the total protein level.The level of Alpha esterase protein was significantly (p < 0.05) increased in the case of Rheum extract (Table 15).Analog results revealed that Citrus limetta seed oil disturbed the defensive and target enzymes of Rhipicephalus microplus via reducing the levels of SOD, GST, MAO, and AChE and increasing NOS level in ticks when compared with the control group 95 .Furthermore, azadirachtin significantly inhibited the activity of AChE when compared with control of N. lugens 96 .Essential oils can diminish esterase, glutathione S-transferases (GSTs) activities, and the total carbohydrate, lipid, and protein contents in Tribolium castaneum 101,102 .
Aloe and Rheum extracts in this study significantly decreased the total protein as compared to the control group.Similarly, reduction in total protein content (representing defensive and target enzymes) is a common occurrence in insects after treatment with toxic compounds, but increased after treatment of a field strain of the mosquito, Culex pipiens, with some IGRs like lufenuron and novaluron 100 .Such variation might be due to using different species and compounds.
In this study, there was an increased level of AChE after treatment with A. vera ethanol extract but decreased PT with its nanoemulsion and R. rhabarbarum extracts.Similarly, essential oils as well as carvacrol have an AChE inhibitory effect indicating that the position of the hydroxyl group in the structure plays an important role 85 .AChE is the main physiological target of many synthetic acaricides such as organophosphate (OP) and carbamate leading to overstimulation of the neurons and rapid twitching of the muscles, convulsions, and death.Treatment with A. vera extract significantly increased AChE level in this work.A parallel study indicated that imidacloprid significantly increased AChE, Glutathione-S-transferases, GSTs, and activities per protein content of the water flea Daphnia magna (Daphniidae: Anomopoda) 21 days PT 103 .A similar recent study showed elevated levels of AChE, α and β esterases, and GSH after treatment of Cx. pipiens with IGRs like lufenuron 100 .
In contrast, AChE level was decreased PT with Rheum nanoemulsion in this study.Alike, AchE activity was significantly inhibited in larvae of R. annulatus PT with fennel oil and its main constituents, trans-anethole and fenchone 104 ; and larvae of Rhipicephalus microplus PT with the n-hexane extract of Calea serrate 105. .Moreover,  93 .
It is worth mentioning that silver and graphene oxide nanoparticles affected insect antioxidant and detoxifying enzymes, inducing oxidative stress and cellular death 106 .The pesticidal action of the synthesized green nanoparticles could be induced because of their penetration into insect exoskeleton, ability to bind to the sulfur element with proteins or DNA phosphorylation, and rapid denaturation of saturation 106,107 .
Plant extracts contain secondary compounds derived from plants that perform useful functions against insects by acting as repellents, antifeedants, and toxins and have provided alternative resources for insect control.They are also characterized by their biodegradation and minimal harmful effects on non-target organisms 19,55,56 .In general, botanicals could be recognized as safe [19][20][21] .R. rhabarbarum is a medicinal edible plant consumed worldwide 59 .A. vera has anticoccidial, antibacterial, and immunomodulatory effects; therefore, it enhances the intestinal health and performance of birds when used as a safe feed additive 108 .The aqueous and hydroethanolic extracts of Tabernaemontana elegans; Calpurnia aurea, Schkuhria pinnata, and Aloe rupestris (leaves, stems, whole plant, and leaves, respectively) not only effectively controlled R. turanicus, but also were safe or very safe on human Vero kidney and liver HepG2 cells 89 .Temulawak (Curcuma xanthorrhiza Roxb) nanoemulsion is safe and improves chickens' productivity and performance and could prevent the risks of antibiotic residues and resistance 109 .No symptoms of skin irritation or abnormal health observation were observed among operators as well as birds 44 and buffaloes 36 , post spraying and pour-on applications, respectively, of essential oils; rabbits PT with aqueous neem extract 14 ; and cattle after spot-on application with aqueous and silver nanoformulations of C. molmol and Z. officinale 10 .

Conclusions
This investigation proved that the novel ethanol extracts of A. vera and R. rhabarbarum and their nanoemulsion induced effective acaricidal and growth-regulating effects against H. dromedarii.Nanoformulations of A. vera and R. rhabarbarum enhanced the efficacy of the ethanol extracts 1.5-2.5 times three days PT, and 1-7 times five days PT and accelerated the speed of killing ticks 2-4 times faster than the ethanol extracts.They also adversely affected the reproductive potential of engorged females.Consequently, they could prevent tick bites and their associated diseases as eco-friendly acaricides.It is indicated from this investigation that botanicals could be used for the progress of benign and eco-friendly acaricides against H. dromedarii.Further studies could be directed towards in vivo and ecotoxicological studies of A. vera and R. rhabarbarum.
Relative toxicity = LC 50 (or LC 90 ) of the least toxic plant extract/LC 50 (or LC 90 ) of each tested plant extract.Times potency = LT 50 of the least toxic plant extract/LT 50 of each tested plant extract Toxicity index = LC 50 of the most toxic plant extract × 100/LC 50 of each tested plant extract.

Table 1 .
Standard polyphenols used for The High-Performance Liquid Chromatography (HPLC) analyses.

Table 2 .
The High-Performance Liquid Chromatography (HPLC) of Aloe vera.

Table 7 .
Toxicity of ethanol plant extracts of Aloe vera and Rheum rhabarbarum against Hyalomma dromedarii males, five days post-treatment.Concentration% Relative toxicity = LC 50 of the least toxic compound/LC 50 of the tested compound.Toxicity index = LC 50 of the most toxic compound × 100/LC 50 of the tested compound.

Table 9 .
The insect growth regulating the effect of plant extracts against Hyalomma dromedarii engorged females.

Table 10 .
82fect of the nanoemulsion of plant extracts post-treatment of Hyalomma dromedarii males.Means followed by the same letter in the same column are not significantly different by ANOVA (p > 0:05).CsNPs encapsulated A. vera gel extract against M. domestica was documented and their relative efficacies were almost 40.65 and 148.51 times more effective than the A. vera crude extrac82.A similar study revealed the acaricidal effect of the aqueous extracts of C. molmol and Z. officinale against H. dromedarii in Egypt as their MO% reached 96 and 84.01%, respectively, 15 days PT with 17%, whereas complete MO was reached seven and nine days PT with 12% of their corresponding AgNPs extracts, synthesized physically via laser ablation.Their LC 50 values reached 10.37, 12.81, 2.38, and 4.12%, respectively, three days PT and their LT 50 values were 5.6, 6.73, 2.25, and 3.56 days, respectively, PT with 4%.Such extracts reduced R. (Boophilus) microplus three days PT of naturally infested cattle by 54.45, 45.73, 100, and 100%, respectively, whereas such ticks acquired resistance against Deltamethrin (Butox®) Vol:.(1234567890) Scientific Reports | (2023) 13:16802 | https://doi.org/10.1038/s41598-023-43776-6www.nature.com/scientificreports/and

Table 11 .
Toxicity of nanoemulsions of Rheum rhabarbarum and Aloe vera post-treatment of Hyalomma dromedarii males three days post-treatment.Concentration: Relative toxicity = LC 50 of the least toxic compound/LC 50 of the tested compound.Toxicity index = LC 50 of the most toxic compound × 100/LC 50 of the tested compound.

Table 12 .
Toxicity of the tested nanoemulsion from Aloe vera and Rheum rhabarbarum five days posttreatment of Hyalomma dromedarii males.Concentration: %; df: degree of freedom.Relative toxicity = LC 50 of the least toxic compound/LC 50 of the tested compound.Toxicity index = LC 50 of the most toxic compound × 100/LC 50 of the tested compound.

Insect growth regulating effects of nanoemulsions against H. dromedarii
This study illustrated the significant (p < 0.05) adverse effect of nanoemulsions of A. vera and R. rhabarbarum as IGRs on the reproduction of the engorged females.After treatment with their lowest concentrations (0.75%), the hatchability%, the number of hatched eggs, and the weights of engorged females and egg masses were 61.9%, 116.66, 51.33, and 2.86 g as well as 51.9%, 138.33, 51.0, and 3.93 gm, respectively.Whereas PT with the highest concentration, 25%, egg production was suppressed and engorged female weights were 48.50 and 51.50g, respectively.Nanoemulsions of both extracts adversely affected the reproductive potential of treated engorged females than those of the crude ethanol extracts (Table

Table 13 .
Lethal time (LT) values (per day) and time potency of nanoemulsions of Rheum rhabarbarum and Aloe vera post-treatment of Hyalomma dromedarii males.Times potency = LT 50 of the least toxic plant extract/ LT 50 of each tested plant extract.

Table 14 .
The insect growth regulating effect of nanoemulsion extracts against Hyalomma dromedarii engorged females.Means followed by the same letter in the same column were not significantly different by ANOVA (p > 0:05).

Table 15 .
Biochemical parameters after treatment of male Hyalomma dromedarii with plant extract.Means followed by the same letter on the same raw were not significantly different by ANOVA (p > 0:05).