606 Pakistan Veterinary Journal ISSN: 0253-8318 (PRINT), 2074-7764 (ONLINE) DOI: 10.29261/pakvetj/2023.054 Assessment of Repellency and Acaricidal Potential of Nigella sativa Essential Oil Using Rhipicephalus microplus Ticks Nawal Al-Hoshani1, Muhammad Arfan Zaman2, Khalid M. Al Syaad3, Muhammad Salman*4, Tauseef ur Rehman5 and A Sonia Olmeda6 1Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia; 2Department of Pathobiology, College of Veterinary and Animal Sciences Jhang, Pakistan. 3Department of Biology, College of Science, King Khalid University, Abha 61413, Saudi Arabia. 4Department of Parasitology, University of Agriculture, Faisalabad, 38040, Pakistan; 5Department of Parasitology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Pakistan; 6Animal Health Department, Veterinary Medicine School, Complutense University of Madrid. Av. Puerta de Hierro s/n, 28040 Madrid, Spain *Corresponding author: msalmanhameed@gmail.com ARTICLE HISTORY (23-114) A B S T R A C T Received: Revised: Accepted: Published online: Apil 02, 2023 July 03, 2023 July 11, 2023 July 27, 2023 Owing to the development of resistance in ticks, the presence of drug residues in food products and the non-target toxicity associated with synthetic acaricides, the scientists are forced to discover some other effective tick control alternatives like botanicals. Hence, in this perspective, the current study was focused on the investigation of repellent and acaricidal potential of Nigella (N.) sativa essential oil against the Rhipicephalus (R.) microplus ticks. Moreover, this research also included the phytochemical analysis of N. sativa essential oil through GC-FID procedure which indicated nerol to be its major constituent. Both the repellent and the acaricidal experiments were conducted using the N. sativa essential oil at 1, 2.5, 5, 10 and 20% v/v dilutions. The results of these experiments indicated the N. sativa essential oil to exert repellent, acaricidal and reproductive effects in terms of various parameters with dose-dependent responses. Thus, the N. sativa essential oil may serve as an effective alternative for the control of R. microplus tick infestation. Key words: Acaricidal Essential Oil Nigella sativa Repellent Rhipicephalus microplus Ticks To Cite This Article: Al-Hoshani N, Zaman MA, Al Syaad KM, Salman M, Rehman TU and Olmeda AS, 2023. Assessment of repellency and acaricidal potential of nigella sativa essential oil using rhipicephalus microplus ticks. Pak Vet J, 43(3): 606-610. http://dx.doi.org/10.29261/pakvetj/2023.054 INTRODUCTION Ticks and ticks-borne diseases in animals, particularly the large ruminants, proves to be the most important limiting factor for the livestock economy in developing countries (Basit et al., 2021; Atif et al., 2022; Masih et al., 2022). Various estimates put 80% population of the cattle worldwide to be at risk of being affected with ticks and ticks-borne diseases (Burrow et al., 2019). Ticks as the hematophagous ectoparasites are found globally causing huge financial losses. Among the various genera affecting the ruminants, Rhipicephalus is the most important (Ceylan et al., 2021; Rooman et al., 2021). The species of this genus inflict heavy losses in the form of reduced production either through direct means or indirectly through transmission of various diseases (Hussain et al., 2021; Salman et al., 2023). It can be understood from the fact that Rhipicephalus (R.) microplus alone causes 22-30 billion dollars loss globally per annum (Lew-Tabor and Valle, 2016). Moreover, an engorged female tick of R. microplus is estimated to reduce 0.6g weight in beef calves (Zaman et al., 2012). Synthetic drugs have long served as the epicentre for all the efforts aimed at the prophylactic control and eradication of these ticks through the provision of a quick and an effective response (Selles et al., 2021). However, owing to the long-term irrational use, the ticks have become resistant to most of the available acaricidal drugs (Abbas et al., 2014a; 2014b; Sindhu et al., 2022). Additionally, the detection of residues of these drugs in food products and their non-target toxicity have created serious concerns (Nath et al., 2018; Goswami et al., 2022; Salman et al., 2022). Hence, the vaccination is developed as a preventive strategy against tick infestation, but it also has limited effectiveness. This is because of the presence of different species and the antigenic variations between different strains as a single vaccine cannot protect against infestation of all the species. Moreover, vaccine development is also an RESEARCH ARTICLE mailto:msalmanhameed@gmail.com http://pvj.com.pk/pdf-files/23-114.pdf Pak Vet J, 2023, 43(3): 606-610. 607 expensive process which lowers its practical significance (de La Fuente and Estrada-Pena, 2019). Hence, in the light of above discussion, other alternatives are needed which can provide an effective control of parasites including ticks (Abbas et al., 2014; Zaheer et al., 2021; Bajwa et al., 2022; Jamil et al., 2022; Zaheer et al., 2022a; 2022b). These alternatives may be the botanicals as almost 2000 species of plants are reported to have pest control properties (Abbas et al., 2018). These properties may be exhibited by the plants as whole or their components (Mamun and Ahmed, 2011; Salman et al., 2020). Many experiments conducted on essential oils extracted from several plants for the investigation of their acaricidal and repellent effects against various genera of ticks like Rhipicephalus, Amblyomma, Hyalomma and Dermacentor have shown promising results (Benelli and Pavela, 2018). Similarly, the N. sativa essential oil is also reported to have insecticidal, acaricidal and repellent properties against a number of arthropods. It has shown its effectiveness even against the R. annulatus ticks (Aboelhadid et al., 2016; Carroll et al., 2016; Faheem and Abduraheem, 2019; Ndirangu et al., 2020). However, the repellent and acaricidal effects of N. sativa essential oil against the R. microplus ticks have not yet been researched. Therefore, the current experiment was aimed at the investigation of both the acaricidal and the repellent effects of N. sativa essential oil using the R. microplus ticks from large ruminants. MATERIALS AND METHODS Ticks collection and identification: Ticks were collected from the naturally-infested cattle and buffaloes in the Faisalabad district of Punjab, Pakistan during the summer season. The collections were made from those animals which were not administered any acaricidal drug during the last 30 days. These ticks were collected in plastic jars having minute holes in their lids and water- soaked cotton swabs placed inside for the provision of proper aeration and humidity. After collection, these ticks were brought to the Chemotherapy Laboratory, Department of Parasitology, University of Agriculture Faisalabad, Pakistan. After washing and drying, they were subjected to the identification process under the 10X magnification of stereomicroscope using the reference guide of Walker (2003). Essential oil extraction and analysis: For the extraction of essential oil, the N. sativa seeds were ground and subjected to the hydro-distillation process using Clevenger apparatus. The essential oil, thus obtained, was processed through the GC-FID (Gas Chromatography-Flame Ionization Detection) procedure at Central Hi-Tech Laboratory, University of Agriculture Faisalabad for the determination of its chemical composition. The Shimadzu Gas Chromatograph (GC- 17A) apparatus coupled with a flame ionization detector having DB WEX column (30m×0.25) with flow rate of 20 ml/min for its nitrogen mobile phase was used for this purpose. While processing, the oven temperature adjustments were followed as 90, 180 and 240°C for 2, 2 and 3 minutes respectively whereas the injector and the detector temperatures were respectively set at 250 and 270°C. During this procedure, the constituent components of the N. sativa essential oils were identified by comparing the retention times of the standards and those of the sample (Belhachemi et al., 2022). Preparation of dilutions: The essential oil of N. sativa was tested at 1, 2.5, 5, 10 and 20% v/v dilutions of the acetone solvent. The obtained results were then compared to those of the positive and negative control groups. The positive control groups comprised of DEET (diethyltoluamide) and cypermethrin for the repellency and the acaricidal treatments respectively. However, acetone acted as negative control treatment for both the experiments. Repellency experiment Climbing test: Tick climbing test was conducted following the protocol described by Ndungu et al. (1995). For this experiment, 10 ticks were observed for the estimation of repellency caused by N. sativa essential oil and the control treatments. The number of ticks above the filter paper for each treatment used in this experiment were used for the calculation of percent repellency. During the experiment, a temperature of 27°C and a relative humidity of 80% was maintained. The following formula was used the calculation of percent repellency: Where C and T represented the number of counted ticks above the filter paper for the control and the sample treatment respectively. Acaricidal experiment Dipping test: Following the procedure of Koc et al. (2013), ten adult R. microplus ticks were dipped for 5 minutes in each dilution. Then they were put into the jars and placed inside the biological oxygen demand (BOD) incubator at 90% humidity and 27°C for 24 hours. After this, the ticks were examined and the mortality was calculated. During this experiment, each dilution was tested in three replications. Percent mortality was determined using the following formula (Sousa et al., 2022): Adult immersion test: For this test, the protocols of Drummond et al. (1973) were observed and ten engorged female R. microplus ticks were immersed in each of the test dilutions for 30 seconds. After this immersion, the ticks were gently dried with the help of tissue paper. Then, they were placed inside the BOD incubator at 27°C and 90% humidity for 20 days till the ovipositing. The eggs, thus collected, were then weighed and again put into the incubator 27°C and 90% humidity for 30 days. This experiment was replicated thrice and the required parameters were calculated using the given formulas (Castro et al., 2018). Pak Vet J, 2023, 43(3): 606-610. 608 Syringe test: Following the procedure of Sindhu et al. (2012), this test was conducted for the calculation of larval mortality (LM). This test started with taking a weighed sample of eggs into specially designed syringes and keeping it in the BOD incubator for hatching. The hatched larvae (14 days old) were used and the mortality was recorded at 24 hours post-treatment with the specified dilutions. While counting, only the walking larvae were considered alive. Following formula was used for the calculation of larval mortality (FAO, 2004): Statistical analysis: The results of various experiments were statistically analysed through ANOVA, Tukey’s Means Comparison Test and Probit Analysis with the help of IBM SPSS software using 95% confidence level and considering the results to be significant when P<0.05 (Barrios et al., 2022; Park et al., 2022). RESULTS AND DISCUSSION Infestation with ticks, especially the R. microplus, is responsible for huge economic damage across the world (Jabeen et al., 2022). Mainly, these tick infestations are kept under control through the use of various synthetic acaricides but the emergence of serious threats have led to the discovery of new means of ticks control such as the essential oils. These essential oils are the secondary byproducts of plants’ metabolism which act through different modes of action for the provision of effective tick control (Salman et al., 2020; Sharmeen et al., 2021). The extraction of essential oils from the plants can be achieved through different techniques yet the N. sativa essential oil was obtained with the help of hydro- distillation. The extracted N. sativa essential oil upon GC- FID analysis was found to contain various components. Among these detected components, nerol was found to be the major component of this essential oil with the highest concentration of 24.2%. This finding resembles the results of a previous study (Marichali et al., 2016) but the difference in the concentration of the detected nerol may be due to the various factors like extraction technique used, soil nature, age of the plant and the type of cultivar (Moghaddam and Mehdizadeh, 2017; Ayub et al., 2023). The components detected in the N. sativa essential oil are listed in Table 1 corresponding to their respective retention times and percent concentrations. Fig. 1: Repellency for Nigella sativa essential oil. A: N. sativa oil 1%; B: N. sativa oil 2.5%; C: N. sativa oil 5%; D: N. sativa oil 10%; E: N. sativa oil 20%; F: Negative control; G: Positive control Bars with same superscript symbols differ non-significantly from each other (P>0.05) Fig. 2: Product Effectiveness Exhibited for Essential Oil of Nigella sativa. A: N. sativa oil 1%; B: N. sativa oil 2.5%; C: N. sativa oil 5%; D: N. sativa oil 10%; E: N. sativa oil 20%; F: Negative control; G: Positive control; Bars with same superscript symbols differ non-significantly from each other (P>0.05). Table 1: Composition of Nigella sativa essential oil Name of the Component Retention Time (min) Concentration (%) Acetaldehyde 2.320 9.3 Geraniol 5.160 6.0 Gamma-undecalactone 7.800 7.5 Isopropyl acetate 13.040 6.8 Octanal 16.560 6.8 Gamma-terpinene 19.360 8.3 Benzaldehyde 22.560 6.5 Furfuryl alcohol 25.680 4.4 Linalool 28.760 3.8 Limonin 31.080 3.1 Unknown 33.000 6.1 Citral 37.280 6.6 Nerol 40.520 24.2 As the repellency of R. microplus ticks, towards N. sativa essential oil, is concerned, varying dose-dependent responses were observed for different treatments. However, only the 20% dilution of the essential oil was capable of eliciting such response that differed non- significantly (P>0.05) from that of the DEET treatment as shown in Fig. 1. Moreover, the EC50 and the EC90 values as calculated through the Probit Analysis were 8.435 and 27.456% respectively. Pak Vet J, 2023, 43(3): 606-610. 609 Table 2: Mortalities for Nigella sativa essential oil against larvae and adult ticks. Treatment A B C D E F G Larvae 3.00±1.73A 13.00±7.55A 46.33±7.50B 78.67±13.50C 93.67±7.09C 4.67±2.52A 98.00±3.46C Adults 10.00±10.00AB 13.33±5.77AB 36.67±11.55BC 63.33±11.55CD 86.67±15.28DE 6.67±5.77A 96.67±5.77E A: N. sativa oil 1%; B: N. sativa oil 2.5%; C: N. sativa oil 5%; D: N. sativa oil 10%; E: N. sativa oil 20%; F: Negative control; G: Positive control; Mean values (±SD) with same superscript letters within the row differ non-significantly from each other (P>0.05). Table 3: Effect of different treatments on fecundity index and oviposition reduction of adult female ticks. Treatment Fecundity Index Oviposition Reduction (%) Egg Hatchability (%) Reproductive Estimation (×20000) A 52.15±8.44A 2.07±15.85A 80.2±7.2A 42.19±10.44A B 43.68±4.57AB 17.96±8.58AB 76.0±5.3A 33.05±1.21A C 32.56±2.59BC 38.85±4.87BC 59.7±4.9B 19.44±2.28B D 19.73±3.89C 62.94±7.30C 38.5±6.2C 7.76±2.75BC E 5.36±0.91D 89.94±1.70D 17.8±2.8D 0.97±0.29C F 53.25±7.84A 0.00±14.73A 84.1±6.1A 44.50±3.75A G 1.14±0.83D 97.86±1.56D 11.2±5.7D 0.10±0.02C A: N. sativa oil 1%; B: N. sativa oil 2.5%; C: N. sativa oil 5%; D: N. sativa oil 10%; E: N. sativa oil 20%; F: Negative control; G: Positive control; Mean values (±SD) with same superscript letters within the column differ non-significantly from each other (P>0.05). This repellent response of N. sativa essential oil may be ascribed to the nerol and the synergistic action of all the components (Susurluk, 2023). Nerol, being the major component of this essential oil, has proved its repellent efficacy against ticks and mosquitoes in a previous experiment (Wong et al., 2022). These volatile components produce a vapour barrier which imparts them a repellent action and drives these arthropods away from this odorous source. However, this repellent barrier diminishes relatively quickly due to the volatility of these substances (Salman et al., 2020). Similar to the repellent experiment, a dose-dependent acaricidal response was observed for the N. sativa essential oil. From these results, the observation of Ellse and Wall (2014) was confirmed which advocated the higher susceptibility of larvae to the essential oils as compared to adult ticks (Table 2). This acaricidal potential of the N. sativa essential oil may have been exerted by the synergistic action of all its components. Moreover, the extract of N. sativa has already proven its acaricidal efficiency (Aboelhadid et al., 2016; Soares et al., 2016). Furthermore, the N. sativa essential oil not only exerted its acaricidal effect in terms of mortality but also influenced the reproductive capability of the R. microplus ticks. This reproductive effect was manifested as the decline in several parameters like oviposition, egg hatchability and overall reproductive performance (Table 3). During the current experiment, the 10% concentration was indicated to have non-significantly different (P>0.05) results from those of the cypermethrin-treated positive control group as shown in Fig. 2. The essential oils exert their reproductive and acaricidal impacts owing to their contact toxicity. They cause break down of cuticular waxes and clog the ticks’ respiratory spiracles resulting in water stress and suffocation (Agwunobi et al., 2020). Moreover, these oils penetrate the cuticle and diffuse into the haemolymph which ultimately transports them to internal organs like salivary glands and ovaries, thus, causing impairment of the digestive and reproductive systems (Remedio et al., 2016; Wang et al., 2020). To add further, the neurotoxic impact of the essential oils may also influence the survival of ticks (Selles et al., 2021). Conclusions: The results of this research indicate N. sativa essential oil to be an effective choice for the control of R. microplus ticks infestation by either repelling or killing them. However, these findings need to be validated through field experiments before recommending its commercial use. Moreover, means of extending the residual life of essential oils as well as the extraction techniques for obtaining maximum oil yield need to be refined. Conflict of Interest: The authors have no conflict of interest. Authors Contribution: NA-H, MAZ, KMAS and MS designed the experiment. MS conducted the research trial. NA-H, KMAS, TUR and ASO provided advisory services throughout the experiment. NA-H, KMAS, TUR and ASO helped in statistical analysis. All authors contributed in writing and approving this manuscript. Acknowledgements: Authors acknowledge the Princess Nourah bint Abdulrahman University Researchers Supporting Project Number (PNURSP2023R437), Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia. The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for supporting this work under the grant number (R.G.P. 2/345/44). REFERENCES Abbas A, Abbas RZ, Khan JA, et al., 2014. Integrated strategies for the control and prevention of dengue vectors with particular reference to Aedes aegypti. Pak Vet J 34:1-10. Abbas A, Abbas RZ, Masood S, et al., 2018. Acaricidal and insecticidal effects of essential oils against ectoparasites of veterinary importance. Bol Latinoam Caribe Plantas Med Aromát 17:441-52. Abbas RZ, Colwell DD, Iqbal Z, et al., 2014b. Acaricidal drug resistance in poultry red mite (Dermanyssus gallinae) and approaches to its management. World Poult Sci J 70:113-24. Abbas RZ, Zaman MA, Colwell DD, et al., 2014a. Acaricide resistance in cattle ticks and approaches to its management: the state of play. Vet Parasitol 203:6-20. Aboelhadid SM, Mahran HA, El-Hariri HM, et al., 2016. Rhipicephalus annulatus (Acari: Ixodidae) control by Nigella sativa, thyme and spinosad preparations. J Arthropod Borne Dis 10:148. Agwunobi DO, Hu Y, Yu Z, et al., 2020. Cymbopogon citratus essential oil–induced ultrastructural & morphological changes in the midgut, cuticle & Haller's organ of the tick Haemaphysalis longicornis (Acari: Ixodidae). Syst Appl Acarol 25:2047-62. Atif FA, Abbas RZ, Mehnaz S, et al., 2022. First report on molecular surveillance based on duplex detection of Anaplasma marginale and Theileria annulata in dairy cattle from Punjab, Pakistan. Trop Anim Health Prod 54:155. Pak Vet J, 2023, 43(3): 606-610. 610 Ayub MA, Goksen G, Fatima A, et al., 2023. Comparison of conventional extraction techniques with superheated steam distillation on chemical characterization and biological activities of Syzygium aromaticum L. essential oil. Separations 10:27. Bajwa HUR, Khan MK, Abbas Z, et al., 2022. Nanoparticles: synthesis and their role as potential drug candidates for the treatment of parasitic diseases. Life 12:750. Barrios H, Flores B, Düttmann C, et al., 2022. In vitro acaricidal activity of Piper tuberculatum against Rhipicephalus (Boophilus) microplus. Int J Acarol 48:187-91. Basit MA, Ijaz M, Khan JA, et al., 2021. Molecular evidence and hematological profile of bovines naturally infected with Ehrlichiosis in southern Punjab, Pakistan. Acta Parasitol 67:72-8. Belhachemi A, Maatoug MH and Canela-Garayoa R, 2022. GC–MS and GC–FID analyses of the essential oil of Eucalyptus camaldulensis grown under greenhouses differentiated by the LDPE cover–films. Ind Crops Prod 178:114606. Benelli G and Pavela R, 2018. Repellence of essential oils and selected compounds against ticks—a systematic review. Acta Trop 179:47- 54. Burrow HM, Mans BJ, Cardoso FF, et al., 2019. Towards a new phenotype for tick resistance in beef and dairy cattle: A review. Anim Prod Sci 59:1401-27. Carroll JF, Babish JG, Pacioretty LM, et al., 2016. Repellency to ticks (Acari: Ixodidae) of extracts of Nigella sativa (Ranunculaceae) and the anti-inflammatory DogsBestFriend™. Exp Appl Acarol 70:89- 97. Castro KNDC, Canuto KM, Brito EDS, et al., 2018. In vitro efficacy of essential oils with different concentrations of 1,8–cineole against Rhipicephalus (Boophilus) microplus. Rev Bras Parasitol Vet 27:203- 10. Ceylan O, Uslu A, Ceylan C, et al., 2021. Predominancy of Rhipicephalus turanicus in tick-infested sheep from Turkey: a large-scale survey. Pak Vet J 41:429-33. De La Fuente J and Estrada–Peña A, 2019. Why new vaccines for the control of ectoparasite vectors have not been registered and commercialized? Vaccines 7:75. Drummond REA, Ernst SE, Trevino JL, et al., 1973. Boophilus annulatus and B. microplus: laboratory tests of insecticides. J Econ Entomol 66:130-33. Ellse L and Wall R, 2014. The use of essential oils in veterinary ectoparasite control: a review. Med Vet Entomol 28:233-43. Faheem F and Abduraheem K, 2019. Repellent activity of Nigella sativa, Syzygium aromaticum and Azadirachta indica essential oils against the skin and skin product pest (Anthrenus verbasci) in museums. J Innov Pharm Biol Sci 6:57-69. FAO, 2004. Guidelines resistance management and integrated parasite control in ruminants. Food and Agriculture Organization of the United Nations Rome, pp:25-77. Goswami R, Arora N, Mrigesh M, et al., 2022. Effect of eucalyptus oil against tick infestation in cattle. Pharm Innov J 11:804-7. Hussain S, Saqib M, Ashfaq K, et al., 2021. First molecular evidence of Coxiella burnetii in ticks collected from dromedary camels in Punjab, Pakistan. Pak Vet J 42:276-80. Jabeen F, Mushtaq M, Qayyum M, et al., 2022. Tick taxonomy and nucleotide sequence analysis by internal transcribed spacer 2 (ITS 2) in large ruminants of Pothohar, Pakistan. Pak Vet J 42:554-60. Jamil M, Aleem MT, Shaukat A, et al., 2022. Medicinal plants as an alternative to control poultry parasitic diseases. Life 12:449. Koc S, Oz E, Cinbilgel I, et al., 2013. Acaricidal activity of Origanum bilgeri PH Davis (Lamiaceae) essential oil and its major component, carvacrol against adults Rhipicephalus turanicus (Acari: Ixodidae). Vet Parasitol 193:316-9. Lew–Tabor AE and Valle MR, 2016. A review of reverse vaccinology approaches for the development of vaccines against ticks and tick borne diseases. Ticks Ticks Borne Dis 7:573-85. Mamun MSA and Ahmed M, 2011. Prospect of indigenous plant extracts in tea pest management. Int J Agric Res Innov Technol 1:16-23. Marichali A, Dallali S, Ouerghemmi S, et al., 2016. Responses of Nigella sativa L. to zinc excess: focus on germination, growth, yield and yield components, lipid and terpene metabolism, and total phenolics and antioxidant activities. J Agri Food Chem 64:1664-75. Masih A, Rafique A, Siddique AB, et al., 2022. Babesia bovis in large ruminants in Pakistan-molecular detection and haemato- biochemical alterations. Acta Sci Vet 50:1890. Moghaddam M and Mehdizadeh, L, 2017. Chemistry of essential oils and factors influencing their constituents. In: Soft Chemistry and Food Fermentation: Academic Press, pp:379-419. Nath S, Mandal S, Pal S, et al., 2018. Impact and management of acaricide resistance–pertaining to sustainable control of ticks. Int J Livest Res 8:46. Ndirangu EG, Opiyo S and Ng’ang’a MW, 2020. Chemical composition and repellency of Nigella sativa L. seed essential oil against Anopheles gambiae sensu stricto. Trends Phytochem Res 4:77-84. Ndungu M, Lwande W, Hassanali A, et al., 1995. Cleome monophylla essential oil and its constituents as tick (Rhipicephalus appendiculatus) and maize weevil (Sitophilus zeamais) repellents. Entomol Exp Appl 76:217-22. Park JH, Kim HJ, Wimalasena SHMP, et al., 2022. In vitro repellent efficacy of Pogostemon cablin (Blanco) Benth.[Lamiaceae] essential oil and its nanoemulsion against Haemaphysalis longicornis (Acari: Ixodidae). Int J Acarol 48:466-71. Remedio RN, Nunes PH, Anholeto LA, et al., 2016. Morphological alterations in salivary glands of Rhipicephalus sanguineus ticks (Acari: Ixodidae) exposed to neem seed oil with known azadirachtin concentration. Micron 83:19-31. Rooman M, Assad Y, Tabassum S, et al., 2021. A cross–sectional survey of hard ticks and molecular characterization of Rhipicephalus microplus parasitizing domestic animals of Khyber Pakhtunkhwa, Pakistan. PLoS One 16:e0255138. Salman M, Abbas RZ, Mehmood K, et al., 2022. Assessment of avermectins-induced toxicity in animals. Pharmaceuticals 15:332. Salman M, Abbas RZ, Nawaz MY, et al., 2023. Impact of climate change on ticks and ticks-borne zoonotic diseases. In: One Health Triad: Vol 3: Agents (Aguilar-Marcelino L, Younus M, Khan A, Saeed NM and Abbas RZ, eds): Unique Scientific Publishers, Faisalabad, Pakistan, pp:28-33. Salman M, Abbas RZ, Israr M, et al., 2020. Repellent and acaricidal activity of essential oils and their components against Rhipicephalus ticks in cattle. Vet Parasitol 283:109178. Selles SMA, Kouidri M, González MG, et al., 2021. Acaricidal and repellent effects of essential oils against ticks: a review. Pathogens 10:1379. Sharmeen JB, Mahomoodally FM, Zengin G, et al., 2021. Essential oils as natural sources of fragrance compounds for cosmetics and cosmeceuticals. Molecules 26:666. Sindhu ZUD, Naseer MU, Raza A, et al., 2022. Resistance to cypermethrin is widespread in cattle ticks (Rhipicephalus microplus) in the province of Punjab, Pakistan: in vitro diagnosis of acaricide resistance. Pathogens 11:1293. Sindhu ZUD, Jonsson NN and Iqbal Z, 2012. Syringe test (modified larval immersion test): a new bioassay for testing acaricidal activity of plant extracts against Rhipicephalus microplus. Vet Parasitol 188:362-7. Soares AMDS, Penha TA, Araújo SAD, et al., 2016. Assessment of different Lippia sidoides genotypes regarding their acaricidal activity against Rhipicephalus (Boophilus) microplus. Rev Bras Parasitol Vet 25:401-6. Sousa ABBD, Bianchi D, Santos EM, et al., 2022. First description of acaricide resistance in populations of Rhipicephalus microplus tick from the lower Amazon, Brazil. Animals 12:2931. Susurluk H, 2023. Potential use of essential oils from Origanum vulgare and Syzygium aromaticum to control Tetranychus urticae Koch (Acari: Tetranychidae) on two host plant species. PeerJ 11:e14475. Walker AR, 2003. Ticks of domestic animals in Africa: a guide to identification of species. Edinburgh: Bioscience Reports, pp:3-210. Wang M, Hu Y, Li M, et al., 2020. A proteomics analysis of the ovarian development in females of Haemaphysalis longicornis. Exp Appl Acarol 80:289-309. Wong C, Corona C and Coats J, 2022. Biorational compounds as effective arthropod repellents against mosquitoes and ticks. In: Advances in Arthropod Repellents: Academic Press, pp:33-48. Zaheer T, Ali MM, Abbas RZ, et al., 2022b. Insights into nanopesticides for ticks: the superbugs of livestock. Oxid Med Cell Longev 2022:7411481. Zaheer T, Imran M, Pal K, et al., 2021. Synthesis, characterization and acaricidal activity of green-mediated ZnO nanoparticles against Hyalomma ticks. J Mol Struct 1227:129652. Zaheer T, Kandeel M, Abbas RZ, et al., 2022a. Acaricidal potential and ecotoxicity of metallic nano-pesticides used against the major life stages of Hyalomma ticks. Life 12:977. Zaman MA, Iqbal Z, Abbas RZ, et al., 2012. In vitro and in vivo acaricidal activity of a herbal extract. Vet Parasitol 186:431-6.