J Arthropod-Borne Dis, June 2016, 10(2): 148–158 SM Aboelhadid et al.: Rhipicephalus annulatus … 148 http://jad.tums.ac.ir Published Online: January 05, 2016 Original Article Rhipicephalus annulatus (Acari: Ixodidae) Control by Nigella sativa, Thyme and Spinosad Preparations *Shawky Mohamed Aboelhadid 1, Hesham A Mahran 2, Hazem M El-Hariri 3, Khalid Mohamed Shokier 4 1Department of Parasitology, Faculty of Veterinary Medicine, Beni-Suef University, Egypt 2Department of Hygiene, Management and Zoonoses, Faculty of Veterinary Medicine, Beni-Suef University, Egypt 3Department of Community Medicine Research, National Research Center, Egypt 4 Animal Health Research, Beni-Suef Branch, Beni-Suef, Egypt (Received 7 May 2014; accepted 2 Nov 2014) Abstract Background: Several compounds obtained from plants have potential insecticidal, growth deterrent or repellent characteristics. The control of hard ticks by non-chemical substances was targeted in this study. Methods: The effect of 36 materials on in-vitro ticks was studied, including 2 absolute controls (water only or ab- solute ethyl alcohol only), 6 conventionally used spinosad preparations (aqueous solutions), 12 Nigella sativa (N. sativa) preparations (aqueous and alcoholic solutions), and 12 Thyme preparations (aqueous and alcoholic solutions). The engorged ticks were tested in-vitro for mortality and oviposition ability using the studied materials. Results: The final mortality after 48 hours of application in N. sativa aqueous preparations began from 10.0% con- centration, 1.0% to 100% by concentration preparations ≥10%. In addition, N. sativa alcoholic preparations began from 50.0% concentration, 2 % to 100% by concentration ≥5%. Meanwhile, Thyme aqueous and alcoholic prepa- rations began from 70.0% concentration, 5% to 90% by concentration 10–20%. Additionally, spinosad aqueous preparations and both of control preparations (Water and Alcohol) resulted in no mortality. All differences were sta- tistically significant. The oviposition was stopped in N. sativa (aqueous ≥10% and alcoholic ≥5%) and in spinosad (aqueous≥25%). The aqoues dilution of the used matters killed B. annulatus larvae beginning from the concentration 5%. Conclusion: Nigella sativa alcohol 20% was the best of studied preparations being the lowest concentration (20%) that could achieve the highest lethal (100%) effect in shortest time (12 hours). Moreover, Thyme oil and spinosad could not kill 100% of adult but did on larvae. Keywords: Rhipicephalus annulatus, Nigella sativa, Thyme oils Introduction Rhipicephalus annulatus (formerly Boo- philus annulatus) represents one of the main risks for the cattle industry in Egypt. Tick control is more difficult due to the presence of resistant populations to all major families of acaricides (Rodriguez-Vivas et al. 2006). Chemical control is a common strategy used in daily life. Synthetic insecticides became the common used forefront of insect-control- ling agents. However, the environmental threat that these chemicals pose, effects on non- target organisms, and the resistance of insect to insecticides have all raised during the last five decades (Wattanachai and Tintanon 1999, Amer and Mehlhorn 2006). There is an in- creasing need to develop insecticides for con- trolling insect, which are more environmen- tally safe and also biodegradable and target- specific against the mosquitoes. Several com- pounds obtained from plants have potential insecticidal, growth deterrent or repellent characteristics (Isman 2006, Pavela 2008). *Corresponding author: Dr Shawky M Aboelhadid, E -mail: drshawky2001@yahoo.com J Arthropod-Borne Dis, June 2016, 10(2): 148–158 SM Aboelhadid et al.: Rhipicephalus annulatus … 149 http://jad.tums.ac.ir Published Online: January 05, 2016 Essential oils, secondary metabolites of the plants, are odor components that can be extracted from plant tissue by water steam distillation or supercritical fluid extraction. Most of them are complex mixtures of mono- and sesquiterpenes and biologically related phenolic compounds. Essential oils and their volatile constituents are commonly used in the prevention and treatment of human ill- nesses. Various essential oils (garlic oil, on- ion oil, etc) have also been documented to exhibit acute toxic effects against insects (Aboelhadid et al. 2013). Nigella sativa is an herbal plant grows in countries of the Mediterranean Sea area, Pa- kistan and India. The seed of it is known as black seed, HabbatuSawda, and Habatul Baraka ‘‘the Blessed Seed’’. It is referred to the prophet Mohammed as having healing pow- ers. The essential oil in its extract was shown to have antihelminthic effect, antinematodal and antischistosomal (Mahmoud et al. 2002). There is no available data about the applica- tion of this oil on ticks. Thyme (Thymus vulgaris L), a perennial labiate, is endemic to the Mediterranean area. Its components and quality vary with ge- ographical distribution, growing states, and stage and method of extraction (Stahl- Biskup and Sáez 2002). The biological ac- tivity of Thyme was found to have insecti- cide potential of obtained extracts using both organic solvents and hydro-distillation (Regnault-Roger et al. 1993, Isman et al. 2001, Knio et al. 2008). Some essential T. vulgaris chemotypes or the essential oils obtained from them had larvicidal and adulticidal efficien- cy. There is no previous work to investigate its effect on ticks. Spinosad was first registered for agricul- tural/horticultural use in the late 1990s and by 1999; it was approved for use on over 100 crops in 24 different countries (Thomp- son et al. 2000). Being a natural product de- rived from fermentation of the microorgan- ism Saccharopolyspora spinosa (Mertz and Yao 1990), spinosad has several favorable characteristics for a pesticide. For example, Anastas et al. (1999) reported that spinosad will not bio-accumulate volatize or persist in the environment and will degrade naturally when exposed to light. In addition, spinosad displays activity against a range of insect pests, especially those in the genera of Lepi- doptera, Diptera and Thysanoptera, and to a lesser extent the Coleoptera and Orthoptera (Thompsonet et al. 2000). Spinosad gives highly efficacious control of many caterpil- lars of lepidopteran insects on numerous crops, including cotton, apples, sweet corn, pota- toes, fruiting/leafy vegetables and tobacco, however, Spinosad is relatively non-toxic to most beneficial insects and mites (DeAmicis et al. 1997). The present investigation aimed to evalu- ate the acaricidal effect of N. sativa and Thyme oils and also spinosad against adult and larvae of R. annulatus through using dif- ferent dilutions and different solvents. Materials and Methods Sampling of R. annulatus In summer 2012, engorged females ticks were carefully detached from naturally in- fested cattle according to Walker et al. (2003). The collected ticks were kept alive in a special glass tubes closed with hydro- phobic cotton a strip of filter paper soaked in 15 % NaCl. In the laboratory of Parasitology, Faculty of Veterinary Medicine, Beni Suef University, the tubes were labeled, incubated at 28 ˚C and relative humidity of 85 % for further studies. Procedure of biological studies Each engorged female tick was put sepa- rately in glass tube contains a filter paper soaked in 15% NaCl, was put to give a rela- tive humidity of 85–90% (BOD incubator, FelpR), then the tubes were closed tightly with a cotton tampon and were labeled (ani- J Arthropod-Borne Dis, June 2016, 10(2): 148–158 SM Aboelhadid et al.: Rhipicephalus annulatus … 150 http://jad.tums.ac.ir Published Online: January 05, 2016 mal host, date, tick species), and were kept in an incubator at the same conditions during the whole period of the experiments. The tubes were observed daily to record the du- ration of the preoviposition and oviposition periods, daily egg output then the eggs were placed in sterile disposable syringes, identi- fied, plugged with cotton balls and were placed in the incubator under the previously mentioned conditions to observe the hatch- ing. The pre-hatching and hatching periods were noticing and recording. Oils extraction from plants The used oils of N. sativa and Thyme were obtained by cold squeezing of the plant seeds. In this work, the oils were obtained from Royal herbal company. Preparation of alcoholic and aqueous so- lutions of the used oils Five concentrations of (1, 3, 5, 10 and 20%) were prepared for oils (N. sativa and thyme), were diluted by distilled water or ethyl alcohol as solvents. The control indi- viduals were treated with ethyl or distilled water only. Preparation of spinosad (TracerR) for ap- plied on adult tick This product was used in agriculture for control of insects. Different concentrations were prepared from this product according the label of the manufacturing (2 ul, 20 ul and 40 ul for 50 ml water). The higher con- centrations 10%, 25 % and 50% were tested. Application methods for treatment of ticks with the used oils and product: Adult immersion test (AIT) Semi and fully engorged females of Rhipicephalus (Boophilus) annulatus, B. annulatus were used in the present study were to adult immersion test (AIT) as de- scribed in Drummond et al. (1973) and Be- navides et al. (1999), the engorged females of R. annulatus, after being washed in a sieve with tap water, were dried on soft absorbent paper. After that, 50 females were divided into five groups of 10 females each and were immersed in Petri dishes containing the above different concentrations of oils, for 2 min. The control group was also composed of 10 engorged females that had been immersed in alcohol or distilled water for the same pe- riod. Ticks were then dried in absorbent pa- per and placed in an incubator at 28 ˚C and 85% relative humidity. The observations of oil effects on tick mortality were recorded at 6, 12, 24 hrs and 48hs post treatment. Evaluation of the acaricidal efficiency of the used materials The mortality rate of treated adults was reported by noticing the number of dead ticks for each concentration. The oviposition ability of the treated engorged females and the ability of these eggs to hatch were inves- tigated. Dead ticks were diagnosed based on three criteria, signs of cuticle darkness and hemorrhagic skin lesions, leg movement, and halted Malpighian tube movement. The legs movement was tested with a paintbrush with ticks placed in an inverted position un- der a stereomicroscope lamp. The change of cuticle color and hemorrhagic skin lesions were demonstrated in dead ticks. Failure in egg laying was then directly observed by eye (Chungsamarnyart and Jansawan 2001). Larval immersion test The different concentrations of the used oils in aqueous solution were screened against the unfed (15 days old) larvae. The larval immersion technique was proposed by Shaw (1966). A group of approximately 600 larvae, 5 replicates in each one 120 larvae for each concentration and control, were distrib- uted on a 14 cm diameter of filter paper ly- ing in a Petri dish. Five milliliters of each concentration was poured over the larvae. Another 14 cm diameter filter paper was J Arthropod-Borne Dis, June 2016, 10(2): 148–158 SM Aboelhadid et al.: Rhipicephalus annulatus … 151 http://jad.tums.ac.ir Published Online: January 05, 2016 placed over the larvae and 5 ml of the extract poured over it. After the immersion period of 5 min, the larvae were removed from the fil- ter paper and were placed in a folded filter paper (5×10 cm) with the openings closed with a tape and then were incubated at 28 ˚C and 85% relative humidity (Borges et al. 2003). Statistical analysis The collected data were coded, tabulated, and statistically analyzed using SPSS program (Statistical Package for Social Sciences) soft- ware version 18.0. The data was independent categorical, descriptive statistics were done as number and percentage, while Inferential analyses were done using Chi square. The level of significance was taken at P< 0.050 is significant, otherwise is non-significant. Results The effect the used materials on adult R. annulatus No lethal effect were noticed in ticks that were subjected to the 2 absolute control (water only and ethanol only) and the 6 convention- ally used Tracer preparations (Aqueous solu- tions of different concentrations, 2 µl/50 ml, 20 µl/50 ml, 40µl/50 ml, 10%, 25% and 50%). None of the studying materials produced any lethal effects 6 hours after application. The highest lethal effect 12 hours after ap- plication was N. sativa aqu (50% and 100%), N. sativa alc (20%, 50% and 100%) this was significantly higher than N. sativa aqu 20% and N. sativa alc 10% that reached 90% death. The final evaluation of different materials is 48 hours after application of each material. The most lethal materials (100% death) were N. sativa aqu (10%, 20%, 50% and 100%), N. sativa alc. (10%, 20%, 50% and 100%), this was significantly higher than the lesser lethal material (90% death) produced by Thyme aqu (10% and 20%) and Thyme alc (10% and 20%), they also significantly higher than the lesser lethal material (70% death) produced by N. sativa aqu 5% and Thyme alc 5%), they also significantly higher than the lesser lethal material (50% death) produced by N. sativa alc 3%), they also significantly higher than the lesser lethal material (10% death) produced by N. sativa aqu 3%, the other ma- terials produced no lethal effects (Table 1). The effect on egg oviposition of R. annulatus The oviposition of live ticks after treat- ment, the live ticks after application of N. sativa oil stopped egg ovipositin in aqu (10%, 20%, 50% and 100%), N. sativa alc (5%, 10%, 20%, 50% and 100%). By fol- lowing the ability of the produced eggs from treated live ticks, the eggs hatched in the ex- pected period of normal eggs from untreated ticks (14 days). The live ticks subjected to different con- centrations of Thyme oils oviposited normal eggs just after application (24 h). These eggs hatched in the mean time after incubation. At the lower concentrations of spinosad (TracerR) the ability of treated ticks to oviposition did not affect and the produced eggs were normal in quantity and time of oviposition. These produced eggs hatched in the expected time of the normal eggs from untreated ticks, while the oviposition activity of treated ticks stopped at the higher con- centrations (25% and 50%) where the ticks became to shrinkage, contrcted but not dead and it did not produced eggs (Table 3). The effect on larvae of R. annulatus No lethal effect was noticed in ticks that were subjected to the absolute control (water only) and N. sativa aqueous 1% and Thyme aqueous 1%. The lethal effect was signifi- cantly variable among different concentra- tions of aqueous concentrations of N. sativa, Thyme and Spinosad, it reached 100% in aqueous concentrations of N. sativa≥5% and Spinosad ≥20µl/50ml while none of aqueous concentrations of Thyme showed that level of mortality (Table3). J Arthropod-Borne Dis, June 2016, 10(2): 148–158 SM Aboelhadid et al.: Rhipicephalus annulatus … 152 http://jad.tums.ac.ir Published Online: January 05, 2016 Table 1. Effect of different materials on Ticks viability S Material 6 hours 12 hours 24 hours 48 hours 1 Water only 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 2 Ethanol only 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 3 N. sativa aqu 1% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 4 N. sativa aqu 3% 0 (0%)a 0 (0%)a 5 (10%)b 5 (10%)b 5 N. sativa aqu 5% 0 (0%)a 25 (50%)c 35 (70%)d 35 (70%)d 6 N. sativa aqu 10% 0 (0%)a 35 (70%)d 45 (90%)e 50 (100%)f 7 N. sativa aqu 20% 0 (0%)a 45 (90%)e 50 (100%)f 50 (100%)f 8 N. sativa aqu 50% 0 (0%)a 50 (100%)f 50 (100%)f 50 (100%)f 9 N. sativa aqu 100% 0 (0%)a 50 (100%)f 50 (100%)f 50 (100%)f 10 N. sativa alc 1% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 11 N. sativa alc 3% 0 (0%)a 5 (10%)b 25 (50%)c 25 (50%)c 12 N. sativa alc 5% 0 (0%)a 25 (50%)c 35 (70%)d 50 (100%)f 13 N. sativa alc 10% 0 (0%)a 45 (90%)e 50 (100%)f 50 (100%)f 14 N. sativa alc 20% 0 (0%)a 50 (100%)f 50 (100%)f 50 (100%)f 15 N. sativa alc 50% 0 (0%)a 50 (100%)f 50 (100%)f 50 (100%)f 16 N. sativa alc 100% 0 (0%)a 50 (100%)f 50 (100%)f 50 (100%)f 17 Thyme aqu 1% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 18 Thyme aqu 3% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 19 Thyme aqu 5% 0 (0%)a 0 (0%)a 25 (50%)c 35 (70%)d 20 Thyme aqu 10% 0 (0%)a 0 (0%)a 40 (80%)d,e 45 (90%)e 21 Thyme aqu 20% 0 (0%)a 0 (0%)a 40 (80%)d,e 45 (90%)e 22 Thyme aqu 50% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 23 Thyme aqu 100% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 24 Thyme alc 1% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 25 Thyme alc 3% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 26 Thyme alc 5% 0 (0%)a 0 (0%)a 25 (50%)c 35 (70%)d 27 Thyme alc 10% 0 (0%)a 0 (0%)a 40 (80%)d,e 45 (90%)e 28 Thyme alc 20% 0 (0%)a 0 (0%)a 40 (80%)d,e 45 (90%)e 29 Thyme alc 50% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 30 Thyme alc 100% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 31 Spinosadaqu µl/50ml 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 32 Spinosad aqu20µl/50ml 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 33 Spinosad aqu40µl/50ml 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 34 Spinosadaqu 10% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 35 Spinosadaqu 25% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a 36 Spinosadaqu 50% 0 (0%)a 0 (0%)a 0 (0%)a 0 (0%)a alc: Alcoholic form, aqu: Aqueous form. %: Percentage of died ticks in relation to studied sample (50) of each group, a,b,c,d,e,f: Homogenous groups so that, similar letters are not-significantly different, while dif- ferent letters are significantly different. J Arthropod-Borne Dis, June 2016, 10(2): 148–158 SM Aboelhadid et al.: Rhipicephalus annulatus … 153 http://jad.tums.ac.ir Published Online: January 05, 2016 Table 2. Effect of different materials on Ticks oviposition ability S Material Oviposition 1 Water only Normal 2 Ethanol only Normal 3 N. sativaaqu 1% Normal 4 N. sativaaqu 3% Normal 5 N. sativaaqu 5% Normal 6 N. sativaaqu 10% Stopped 7 N. sativaaqu 20% Stopped 8 N. sativaaqu 50% Stopped 9 N. sativaaqu 100% Stopped 10 N. sativaalc 1% Normal 11 N. sativaalc 3% Normal 12 N. sativaalc 5% Stopped 13 N. sativaalc 10% Stopped 14 N. sativa alc 20% Stopped 15 N. sativaalc 50% Stopped 16 N. sativaalc 100% Stopped 17 Thyme aqu 1% Normal 18 Thyme aqu 3% Normal 19 Thyme aqu 5% Normal 20 Thyme aqu 10% Normal 21 Thyme aqu 20% Normal 22 Thyme aqu 50% Normal 23 Thyme aqu 100% Normal 24 Thyme alc 1% Normal 25 Thyme alc 3% Normal 26 Thyme alc 5% Normal 27 Thyme alc 10% Normal 28 Thyme alc 20% Normal 29 Thyme alc 50% Normal 30 Thyme alc 100% Normal 31 Spinosadaqu 2µ l/50ml Normal 32 Spinosad aqu20µl/50ml Normal 33 Spinosad aqu40µl/50ml Normal 34 Spinosadaqu 10% Normal 35 Spinosadaqu 25% Stopped 36 Spinosadaqu 50% Stopped Normal means the quantity and time of egg deposition of untreated normal tick. All ticks that were subjected to each material gave the same response (normal/ stopped) oviposition Table 3. Effect of different concentrations of in aqueous solution on tick larvae after 24 h (n=100 lar- vae in 5 replicates) S Material 24 hours 1 Water only 0 (0%)a 2 N. sativa aqu 1% 0 (0%)a 3 N. sativa aqu 3% 338 (67.6%)d 4 N. sativa aqu 5% 500 (100%)g 5 N. sativa aqu 10% 500 (100%)g 6 N. sativa aqu 20% 500 (100%)g 7 N. sativa aqu 50% 500 (100%)g 8 N. sativa aqu 100% 500 (100%)g 9 Thyme aqu 1% 0 (0%)a 10 Thyme aqu 3% 145 (29.0%)b 11 Thyme aqu 5% 251 (50.2%)c 12 Thyme aqu 10% 342 (68.4%)d 13 Thyme aqu 20% 417 (83.4%)e 14 Thyme aqu 50% 426 (85.2%)e, f 15 Thyme aqu 100% 435 (87.0%)e, f 16 Spinosadaqu 2µ l/50ml 446 (89.2%)f 17 Spinosad aqu20µl/50ml 500 (100%)g 18 Spinosad aqu40µl/50ml 500 (100%)g 19 Spinosadaqu 10% 500 (100%)g 20 Spinosadaqu 25% 500 (100%)g 21 Spinosadaqu 50% 500 (100%)g aqu: Aqueous form N: The total number of died larvae %: Percentage of died ticks in relation to studied sample (500) of each group, a,b,c,d,e,f,g: Homogenous groups so that, sim- ilar letters are not-significantly different, while different letters are significantly different. Discussion Most plants contain compounds that they use in preventing attack from phytophagous (plant eating) insects. These compounds fall into several categories, including repellents, feeding deterrents, toxins, and growth regu- lators. It can be grouped into five major groups, nitrogen compounds (primarily alka- loids, terpenoids, phenolics, proteinase inhib- itor, and growth regulators. Although the pri- mary functions of these compounds are de- fense against phytophagous insects, many are also effective against mosquitoes and other J Arthropod-Borne Dis, June 2016, 10(2): 148–158 SM Aboelhadid et al.: Rhipicephalus annulatus … 154 http://jad.tums.ac.ir Published Online: January 05, 2016 biting Diptera, especially those volatile com- ponents released because of herbivory (Pichersky and Gershenzon 2002) These properties encourage the research- ers to investigate the effect of N. sativa oil on R. annulatus. The present results investi- gated the ability of N. sativa oil on the adult females of R. annulatus. Both alcoholic and aqueous or solution of N. sativa killed all ticks after 12 h or 24 post-treatment from the concentration 5% and 10%. The same results were obtained from the higher concentra- tions. Regarding the oviposition of live ticks after treatment, the live ticks after applica- tion of oils were found to oviposite the nor- mal eggs and in the normal manner. By fol- lowing the ability of the produced eggs from treated live ticks, the eggs hatched in the ex- pected period of normal eggs from untreated ticks. The N. sativa oil (NSO) had many activities, antithelmintics, anticestodal effects in children (Akhtar and Riffat 1991), antischistosomal (Mahmoud et al. 2002), Trichenella spiralis (Abu El Ezz 2005), Aspiculuris tetraptera and Hymenolepis nana (Ayaz et al. 2007), These changes were correlated mainly with the ability of NSO to improve liver function and the immunological system of infected mice and to its antioxidant effects (Mahmoud et al. 2002). The protection is also due to the ability of NSO and TQ to reduce the cytoge- netic damage induced by schistosomiasis in- fection (Aboul-Ela 2002). The N. sativa aque- ous extract could be useful in the treatment of Blastocystis hominis (El Wakil 2007). Thymoquinone, the most abundant constitu- ent of black seed essential oil, is the active principle responsible for many of the seed’s beneficial effects (Muhtasib et al. 2006). The seed has diverse chemical composi- tion. They contain amino acids, proteins, carbohydrates, fixed and volatile oils (Khan 1999). Many of the pharmacological activi- ties have been attributed to quinine and thymoquinone (TQ), constituents in the seed. Mahfouz and El-Dakhakhny (1960) were isolate of ‘nigellone’possess antihistaminic properties. El-Fatatry (1975) reported the iso- lation of thymohydroquinone (THQ) from N. sativa seed volatile oil. The medicinal poten- tial of black seed (N. sativa) referred to the presence of 135 nigellimine and nigellicine (ur-Rahman et al. 1985), saponins and crude fiber as well as minerals such as calcium, iron, sodium and potassium. Other constitu- ents of the volatile oil include thymol (Aboutabl et al. 1986). From these constituents of N. sativa oil may be explaining the acaricidal effect of the oil on adult ticks. These components may be cause saphocation of the ticks due to immer- sion in the solutions. It was noticed that the crude Thyme oil (100%) or 50%, 1% and 3% diluted oil by water or alcohol did not kill any ticks. The solvent (water or alcohol) has no effect on the ability of Thyme oil to cause death of the treated ticks. The tick deaths reached to 90% of treated ticks at concentrations of 10% and 20% in 48hs PA. The live ticks from these concentrations oviposited normal eggs just after application by short time (24 h) but the same quantity. These eggs hatched in the mean time after incubation. The insecticidal effect was ensured through the direct toxicity on adult insects and by inhibiting reproduction. Therefore, one can profit using the essential oils of T. vulgaris and T. serpyllumin additionas an effective fumigant against a wide group of agricul- tural pests that damages its host plant. The oils have a toxic effect on adultinsects and inhibit the reproduction through ovicidal and larvicidal effects (Regnaultroger and Hamraoui, 1994). This insecticidal action is also pro- duced by othercomponents of the species such as non-volatile phenols, non-proteinic amino acids, andflavonoids (Regnaultroger and Hamraoui 1995). Thymol was shown to be more potent than Thyme oil as a deterrent factor for reducinegg lying by the mite. Mor- J Arthropod-Borne Dis, June 2016, 10(2): 148–158 SM Aboelhadid et al.: Rhipicephalus annulatus … 155 http://jad.tums.ac.ir Published Online: January 05, 2016 tality percentage reached 100% with both materialsused, however, at low concentra- tions the effect again was more pronounced applyingthymol than applying Thyme oil (El-Gengaihi et al. 1996). Thymus is a multiple constituent's plant its composition groups can categorized into the following groups: essential oil, glycosoides, flavonoids, flavonoid glycosides and pheoliv acids. Most ofthem are complex mixtures of mono- and sesquiterpenesand biologically re- lated phenoliccompounds. Various essential oils have also been documented to exhibit acute toxic effects against internal and exter- nal parasites. Using spinosadin in this study revealed that the low concentrations had no effect on adult R. annulatus. The higher concentra- tions (25 and 50%) led to the adult engorged female tick still alive but did not able to give eggs. Spinosad is primarily a stomach poison with some contact activity and is particularly active against Lepidoptera and Diptera. It is a neurotoxin with a novel mode of action involving the nicotinic acetylcholine recep- tor and apparently the GABA receptors as well (Salgado 1998). The mode of action of the spinosynsis through neural functions dis- ruption, most likely via an alteration of nic- otinic receptor function (Salgado and Saar 2004). Ploch et al. (2007) demonstrated that symptomological assessment of insects treated with spinosad and spinosyn-A has suggested that the most likely mode of action of these insecticides is via a neural mechanism. These studies reported a possibly of a secondary physiological action of spinosyns. The pri- mary interaction was associated with activa- tion of an a-BGT sensitive current, thus sug- gesting a nicotinic mode of action (Crouse et al. 2007). In addition, in some specific in- stances, a high-potency GABAergic response was also noted (Watson 2001). The results presented heredemonstrates that spinosyn-A does not appear to interact with known nico- tinic or GABA target sites. The weak affin- ity of spinosyn-A for displacement of the voltagegated calcium channel radioligand, [3H] VPM is suggestive of an interaction of spinosyns with this channel. Spinosyn J and theC17 pseudoaglycone of spinosyn-A were both less potent than spinosyn-A in the [3H] VPM assay, which is consistent with their reduced biological activity against several insect species (Salgado 2005). In general, the high non-specific binding of these lig- ands makes them suboptimal for studying membrane bound receptors. Recently, how- ever, the utility of one spinosyn ligand, [3H] 5, 6-dihydrospinosyn-A for cloned insect D- a6 receptorshas been demonstrated (Orr et al. 2006). Davey et al. (2001) reported that spraying of 0.05% and 0.15% spinosad on tick-infested cattle achieved 86% and 87% control, respectively, of larval R. annulatus at time of treatment. Besides, Cetin et al. (2009) who demonstrated that spinosad pro- vides a complete control of larval Argus persicus and R. turanicus in a short exposure time. Conclusion Nigella sativa has acaricidal effect than the other used materials. 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