RaTexT®: a novel rapid tick exposure test for detecting acaricide resistance in Rhipicephalus microplus ticks in Brazil.

BACKGROUND: Acaricide resistance in cattle ticks is a significant concern in (sub)tropical regions, particularly Brazil. The Larval Packet Test (LPT) is the standard laboratory bioassay for resistance diagnosis, which requires triplicates of seven acaricidal dilutions plus controls to cover larval mortalities ranging between 0 and 100%. The value of the LPT lies in providing resistance ratios based on the ratio between the LC50 calculated with potentially resistant and susceptible ticks. However, LC50 ratios are difficult to translate into practical advice for farmers. Moreover, LPT requires laboratory facilities to maintain susceptible tick colonies, and it takes 6 weeks to obtain the larvae to be tested by LPT derived from engorged female ticks collected from cattle in the field. Our novel approach was twofold: first, we upgraded the LPT to the Resistance Intensity Test (RIT) by adopting the latest WHO guidelines for resistance detection in mosquitoes, which combines a 1 × recommended dose with 5 × and 10 × concentrated doses to reveal low, moderate and high resistance intensity, respectively. This reduced the number of test papers and tick larvae and, more importantly, provided relevant information on the resistance level. Our second innovative step was to abolish testing larvae entirely and expose partly engorged adult ticks to the same acaricidal doses immediately after removing them from cattle in the field. This resulted in the Rapid Tick exposure Test (RaTexT®), wherein partly engorged adult ticks were exposed to an acaricide-impregnated, specially designed matrix providing test results within 24 h. This approach directly compared resistance detection in tick larvae in the RIT with resistance in adult ticks in RaTexT®. METHODS: Laboratory validation was conducted in Brazil with resistant and susceptible colonies of Rhipicephalus microplus ticks. For field validation, adult R. microplus ticks collected from different cattle farms in Brazil were evaluated for resistance to RaTexT®, and the results regarding their larval progenies were compared with those for the RIT. Partly engorged adult ticks derived from cattle infested with laboratory and field strains of R. microplus were exposed to deltamethrin in RaTexT® containers, which contained six rows of four interconnected compartments, accommodating five to eight semi-engorged female ticks with a preferred size ranging between 5 and 8 mm. The corresponding larvae of each strain were exposed in the RIT to the same deltamethrin concentrations in filter papers. RESULTS: In RaTexT®, mortality in adult ticks from a resistant strain of R. microplus from Seropédica in Brazil was 38.4%, 54.2% and 75.0% at the 1 ×, 5 × and 10 × doses of deltamethrin, respectively. In RIT, mortality of larvae from the same resistant strain was 2.0%, 4.9% and 19.5% at 1 ×, 5 × and 10 × doses, respectively. The results of RaTexT® and RIT agreed since both tests identified a high level of resistance based on a cut-off of 90% mortality. In RaTexT®, mortality of adult ticks from a susceptible strain originating from Porto Alegre was 73.8%, 92.9% and 97.6% at the 1 ×, 5 × and 10 × doses, respectively. In RIT, mortality of larvae from the susceptible strain was 95.2%, 95.2% and 96.8% at the 1 ×, 5 × and 10 × doses, respectively. Interestingly, both tests identified a low number of unexpected resistant individuals in the susceptible strain since the mortality of neither larvae nor adults reached 100%. This effect remained unnoticed in the LPT, wherein a resistance ratio of 159.5 was found based on the LC50 of the resistant strain divided by the LC50 of the susceptible strain. Next, RaTexT® was compared with RIT using adult and larval ticks derived from three field strains of R. microplus in Brazil. RaTexT® detected high levels of resistance to deltamethrin in adult ticks in all strains, which was confirmed in larvae tested by the RIT. Both tests agreed on the same resistance level with significantly lower mortality rates in larvae than in adult ticks. CONCLUSIONS: RaTexT® is a novel rapid pen-site test for detecting acaricide resistance in adult livestock ticks. It potentially replaces laborious tests using larval ticks and provides results within 24 h relevant to acaricide resistance management of livestock ticks.

Selective versus strategic control against Rhipicephalus microplus in cattle: A comparative analysis of efficacy, animal health, productivity, cost, and resistance management.

This study compared selective control versus strategic control against Rhipicephalus (Boophilus) microplus, under conditions of a real dairy farm. Strategic control requires that all animals be treated with acaricide at regular pre-determined intervals. Selective control only requires treatment of infested animals and only when they are at or above a pre-determined threshold. Tick counts on animals and in pasture were performed and the susceptibility of tick populations to the different treatment methods was evaluated at the beginning and end of the study using the Larval Packet Test, Larval Immersion Test, and Adult Immersion Test. Over the four years of the study strategic control was more advantageous than the selective control as the group experienced lower tick burden on animals and in pasture, absence of skin lesions and myiases, lower operating costs, treatments concentrated in fewer months of the year and lower resistance pressure.

Control of ticks and tick-borne diseases in Africa through improved diagnosis and utilisation of data on acaricide resistance.

A meeting, sponsored by the Bill and Melinda Gates Foundation (BMGF) and organised by Clinglobal, was held at The International Livestock Research Institute (ILRI) in Nairobi, Kenya, from 19th - to 21st October 2022. The meeting assembled a unique group of experts on tick control in Africa. Academia, international agencies (FAO and ILRI), the private Animal Health sector and government veterinary services were represented. The significant outcomes included: (i) a shared commitment to standardisation and improvement of acaricide resistance bioassay protocols, particularly the widely used larval packet test (LPT); (ii) development of novel molecular assays for detecting acaricide resistance; (3) creation of platforms for disseminating acaricide resistance data to farmers, veterinary service providers and veterinary authorities to enable more rational evidence-based control of livestock ticks. Implementation of enhanced control will be facilitated by several recently established networks focused on control of parasites in Africa and globally, whose activities were presented at the meeting. These include a newly launched community of practice on management of livestock ticks, coordinated by FAO, an African module of the World Association for the Advancement of Veterinary Parasitology (WAAVP-AN) and the MAHABA (Managing Animal Health and Acaricides for a Better Africa) initiative of Elanco Animal Health.

Resistance to Cypermethrin Is Widespread in Cattle Ticks (Rhipicephalus microplus) in the Province of Punjab, Pakistan: In Vitro Diagnosis of Acaricide Resistance.

Control of the cattle tick Rhipicephalus (R.) microplus mainly relies on chemical acaricides and cypermethrin is the most widely used acaricide in Pakistan. Farmers frequently complain about its low efficacy, thus, the present study was designed to quantify the frequency of cypermethrin resistance in cattle ticks. Engorged female R. microplus were collected and tested for the efficacy of cypermethrin using the FAO-recommended larval packet test. Resistance factors (RF) were estimated at both the lethal concentration for 50% (LC50) and 99% (LC99) of ticks. Thirty-three samples were tested, of which 8/33 (24.24%) were classified as resistant based on the RF50, and all 33 were classified as resistant based on the RF99. In District Sargodha, when only the RF50 was considered, 45.5% of samples were classified as resistant, but at RF99, all tested samples were identified as resistant. In District Okara, the variation in RF50 estimates was 2.2-8.3 and variation in RF99 estimates was 10.6-1139.8. Similar results were found in District Attock, where variations in RF50 were 0.8-8.5 and RF99 ranged from 9-237.3. The study showed that cypermethrin resistance is prevalent in these three districts of Pakistan and is likely to be overestimated by classification based on the RF99.

Acaricide resistance in livestock ticks infesting cattle in Africa: Current status and potential mitigation strategies.

In many African countries, tick control has recently been the responsibility of resource-poor farmers rather than central government veterinary departments. This has led to an increase in acaricide resistance, threatening the welfare of livestock farmers in sub-Saharan Africa. Resistance has evolved to the three classes of acaricides used most extensively in the continent, namely fourth-generation synthetic pyrethroids (SP), organophosphates (OP) and amidines (AM), in virtually all countries in which they have been deployed across the globe. Most current data are derived from research in Australia and Latin America, with the majority of studies on acaricide resistance in Africa performed in South Africa. There is also limited recent research from West Africa and Uganda. These studies confirm that acaricide resistance in cattle ticks is a major problem in Africa. Resistance is most frequently directly assayed in ticks using the larval packet test (LPT) that is endorsed by FAO, but such tests require a specialist tick-rearing laboratory and are relatively time consuming. To date they have only been used on a limited scale in Africa and resistance is often still inferred from tick numbers on animals. Rapid tests for resistance in ticks, would be better than the LPT and are theoretically possible to develop. However, these are not yet available. Resistance can be mitigated through integrated control strategies, comprising a combination of methods, including acaricide class rotation or co-formulations, ethnoveterinary practices, vaccination against ticks and modified land management use by cattle, with the goal of minimising the number of acaricide applications required per year. There are data suggesting that small-scale farmers in Africa are often unaware of the chemical differences between different acaricide brands and use these products at concentrations other than those recommended by the manufacturers, or in incorrect rotations or combinations of the different classes of chemicals on the market. There is an urgent need for a more evidence-based approach to acaricide usage in small-scale livestock systems in Africa, including direct measurements of resistance levels, combined with better education of farmers regarding acaricide products and how they should be deployed for control of livestock ticks.

Determination of the Discriminating Concentration Towards Permethrin for Surveying Resistance in Amblyomma americanum.

Amblyomma americanum Linnaeus (Ixodida: Ixodidae) is ubiquitously present throughout the southeastern United States and is capable of vectoring several pathogens. White-tailed deer are the main host for adult A. americanum. However, this tick species is a generalist that will feed on most vertebrates, including humans, deer, livestock, and pets. Management of this species can be challenging due to a lack of cost-effective strategies. Acaricides are often utilized, however, this may lead to pesticide resistance. The Food and Agriculture Organization of the United Nations (FAO) larval packet test (LPT) was performed on susceptible A. americanum to determine the lethal concentration (LC) and discriminating concentration (DC) values for permethrin. The FAO LPT was used at these pre-established values to compare levels of resistance in ticks collected from a captive deer farm and wild areas representing high and low permethrin exposure settings, respectively. Resistance ratios (RR) calculated from the LC values for the ticks collected from farmed and wild deer ranged between 1 and 2. A. americanum collected from farmed and wild deer were not found to be resistant, however, some samples had slightly elevated RRs as compared to the susceptible laboratory strain, which may suggest tolerance development. Although the A. americanum sampled in this study were not resistant to permethrin, the DC calculated in this study will allow for rapid evaluation of resistance in a permethrin resistance monitoring program such that alternate management strategies can be adopted if resistance is detected.

Variation of diazinon and amitraz susceptibility of Hyalomma marginatum (Acari: Ixodidae) in the Rabat-Sale-Kenitra region of Morocco.

In the present study, the acaricide susceptibility status of Hyalomma marginatum in Morocco was investigated in the Rabat-Sale-Kenitra region using the Larval Packet Test. The overall LC50 value for diazinon and amitraz was 115 ppm (95% CI: [104; 125]) and 22 ppm (95% CI: [21; 23]), respectively. The LC50 values varied significantly between the nine sampled locations (P<0.001) ranging from 75 ppm (95% CI: [65; 84]) in Ouelmes to 179 ppm (95% CI: [139; 201]) in Jorf El Melha for diazinon and from 18 ppm (95% CI: [15; 21]) in Skhirat to 28 ppm (95% CI: [24; 31]) in Ouelmes for amitraz. Sequencing of the target-site of diazinon, acetylcholinesterase 1 (AChE1), indicated that previously reported resistance mutations in AChE1 were absent in ticks from Jorf El Melha surviving 500 ppm diazinon. This study is the first report on the H. marginatum susceptibilty status to the most frequently used acaricides in Morocco and indicates that acaricide tick resistance is emerging.

Acaricidal activity of the hexanic and hydroethanolic extracts of three medicinal plants against southern cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

The southern cattle tick Rhipicephalus microplus is a major problem for the cattle industry in tropical and subtropical areas worldwide. Chemical products are commonly applied to control it; however, their indiscriminate use has resulted in the appearance of resistant lineages. In the last decades, plants have been used as an alternative to conventional acaricidal drugs, as several plant compounds repel activity, decrease the reproductive potential and reduce the survival rate of ticks. For this reason, the in vitro efficacy of hexanic and hydroalcoholic extracts of Randia aculeata, Moringa oleifera and Carica papaya were evaluated against the larvae and engorged females of R. microplus. Larval packet tests and adult immersion tests were performed with seven concentrations of each of the extracts. The extracts obtained with hydroethanolic solution (polar solvent) exhibited a higher acaricidal activity than extracts prepared with n-hexane (non-polar solvent). Hydroethanolic extracts of R. aculeata seed and shell showed the highest larvicidal activity against R. microplus (100 and 91% mortality, respectively) at a concentration of 100 mg/mL. Randia aculeata (seed and shell), M. oleifera and C. papaya treatments at the same concentration (100 mg/mL) also resulted in adult mortality of 85, 75, 66 and 55%, respectively. The adult immersion test showed that hydroethanolic extracts derived from R. aculeata seed significantly reduced the index of egg laying and increased the percentage inhibition of oviposition of female ticks at a concentration of 100 mg/mL. These results indicate that the tested extracts exhibit acaricidal activity and could be considered as potential agents for the development of alternative natural acaricides against R. microplus.

Susceptibility of Ixodes scapularis (Acari: Ixodidae) to Permethrin Under a Long-Term 4-Poster Deer Treatment Area on Shelter Island, NY.

Pesticide resistance in medically significant disease vectors can negatively impact the efficacy of control efforts. Resistance research on ticks has focused primarily on species of veterinary significance that experience relatively high degrees of control pressure. Resistance in tick vectors of medical significance has received little attention, in part because area-wide pesticide applications are not used to control these generalist tick species. One of the few effective methods currently used for area-wide control of medically important ticks, including Ixodes scapularis Say (Acari: Ixodidae), is deployment of 4-poster devices. Deer self-apply a topical acaricide (permethrin) while feeding on corn from the devices. A 4-poster program using permethrin has been deployed on Shelter Island, NY to control I. scapularis populations since 2008. We collected engorged female ticks from deer in this management area and a location in the Mid-Hudson River Valley, NY without area-wide tick control. Larvae were reared from egg masses and their susceptibility to permethrin was tested. Larvae originating from a long-term laboratory colony were used as a susceptible baseline for comparison. Compared against the laboratory colony, resistance ratios at LC-50 for Shelter Island and Hudson Valley I. scapularis were 1.87 and 1.51, respectively. The susceptibilities of the field populations to permethrin were significantly lower than that of the colony ticks. We provide the first data using the larval packet test to establish baseline susceptibility for I. scapularis to permethrin along with information relevant to understanding resistance emergence in tick populations under sustained control pressure from 4-poster devices.

Genotyping of Deltamethrin Resistance in Rhipicephalus (Boophilus) microplus Population in Kerala, South India.

PURPOSE: Genotyping of Rhipicephalus (Boophilus) microplus for polymorphisms in deltamethrin-resistant loci of sodium channel gene by allele-specific PCR (AS-PCR). METHODS: Adult R. (B.) microplus ticks were collected from naturally infested cattle in Kerala. The larval packet test (LPT) was performed with deltamethrin and dose response data were analysed by probit method. Adult and larval tick DNA were amplified by PCR and later sequenced to identify mutations, if any, in the domain II S4-5 linker and domain III S6 regions in para voltage-gated sodium channel gene, at loci that were previously documented to be associated with deltamethrin resistance. Allele-specific PCR was performed for the amplification of target gene locus (C190A and T2134C) to genotype 1000 larvae each, at these loci. Genotype frequency was statistically analysed by Chi-square test. RESULTS: Bioassay using LPT revealed that LC50 and LC95 values of all the R. (B.) microplus isolates in this study were more than double the reported values of reference susceptible strain. Sequence analysis of the PCR amplicons of domain II S4-5 linker of voltage-gated sodium channel gene revealed C190A mutation, A271G mutation as well as A-G mutation at 217th position. AS-PCR done to genotype C190A mutations revealed a frequency of 6%, 15% and 64%, respectively for homozygous-susceptible (SS), heterozygous (RS) and homozygous-resistant (RR) genotypes. In domain III S6 region of the gene, C2121T and A2102T mutations were observed. AS-PCR to genotype the previously reported T2134C mutation revealed 100% SS genotype in R. (B.) microplus isolates of Kerala. CONCLUSIONS: Genotyping of R. (B.) microplus isolates of Kerala for target site mutations reportedly associated with deltamethrin resistance revealed a significantly high frequency of resistant genotypes at II S4-5 linker of voltage-gated sodium channel gene. This study forms the first report of such mutations in Kerala, south India and demands serious attention in the light of increased prevalence of tick-borne haemoparasitic infection.

Rhipicephalus microplus (acari: Ixodidae): Clinical safety and potential control by topical application of cottonseed oil (Gossypium sp.) on cattle.

The present study was performed to determine the acaricidal activity of the cottonseed oil (CSO) against cattle tick Rhipicephalus microplus. CSO was analyzed using Gas Chromatograph with high-resolution Mass Spectrometer (GC-HRMS) to identify the presence of active compounds. In vitro bioassays were performed using larval packet test (LPT) and adult immersion test (AIT) by taking different concentrations of CSO (i.e. 0.1, 0.5, 1.5, 2.5, 5, 7.5, 10 and 12.5%). In vivo acaricidal activity of CSO was evaluated by its topical application on red Sahiwal calves for 144 h. Clinical safety of CSO was evaluated by performing skin irritancy test and examination of hematological profile of calves'. GC-HRMS analysis of CSO revealed the presence of many fatty acids including oleic acid, lauric acid, palmitic acid, stearic acid and other components. Results exhibited that all the concentrations of CSO were effective in reducing the number of ticks and their growth. However, CSO at concentrations of 10% (CSO7) and 12.5% (CSO8) exhibited 100% mortality of R. microplus larvae and adults in LPT and AIT, respectively. In vivo acaricidal assay revealed that CSO7 and CSO8 shown 85% and 89% inhibition of ticks, respectively on calves after 144 h as compared to the control group. CSO was clinically safe on calves' skin with mild erythema up to 20 min. Hematological profile of calves revealed no sign of toxicity after treatment with CSO. Thus, CSO can be used as an alternative and safe drug therapy against R. microplus.

Evaluation of Fipronil Susceptibility in the Lone Star Tick (Acari: Ixodidae).

The lone star tick, Amblyomma americanum Linnaeus, is ubiquitously present in the southeastern United States and will readily parasitize humans and companion animals. Fipronil is the active ingredient in several topically applied products used to manage ticks and fleas on companion animals. Reducing ectoparasite infestations on companion animals decreases the risk that pathogens vectored by these pests are transmitted to these animals and their owners. However, dependence on acaricides can promote the development of resistance. In this study, the Food and Agriculture Organization of the United Nations larval packet test was used to determine the lethal concentration (LC) values and discriminating concentration (DC) for fipronil in the lone star tick. The DC was calculated as 0.02080%. The low magnitude of the DC value suggests that fipronil is an effective active ingredient for A. americanum management. With the LC and DC values determined, emergent resistance can be monitored, potentially allowing for intervention should tolerance develop in A. americanum populations that are in frequent contact with humans and their companion animals.

Molecular detection of Rhipicephalus (Boophilus) annulatus resistance against deltamethrin in middle Egypt.

This study was designed to investigate the susceptibility/resistance of Rhipicephalus (Boophilus) annulatus to commercial deltamethrin 5%. The work was based on in vitro bioassays; adult immersion test (AIT) and larval packet test (LPT). Fully engorged female ticks were collected from 11 locations in Beni-Suef Governorate, Egypt (Alfashin, Eminelaros, Ehnasia, Smusta, Belifia, Aldiabia, Alhalabia, Beshnna, Alkom, Nasr and Beba). Two concentrations of commercial deltamethrin 5% (200 and 400 ppm) were tested by AIT on R. (B.) annulatus. The mortality of ticks at 400 ppm was higher than 200 ppm which was not reached to 100%. Ehnasia tick population was the least susceptible to deltamethrin while Alfashin population was the most susceptible one. Different deltamethrin concentrations (0.000625, 0.00125, 0.0025, 0.005, 0.010 and 0.020) were used in LPT to evaluate the larval mortality. The highest LC50 appeared in Ehanasia tick population (0.01) with 95% confidence interval (0.005 to 0.01) meanwhile, the lowest LC50 appeared in Alfashin (0.0006) with 95%CI (0.0005 to 0.0007). Thus, it is possible to deduce that one population was considered susceptible; seven populations were resistant level I and three populations were resistant level II. Additionally, PCR revealed that the resistant alleles (R) appeared in two localities (Ehnasia and Aldiabia). It was concluded that at least two tick populations of the investigated locations were of possible resistant to deltamethrin.

Toxic and repellent activity of selected monoterpenoids (thymol, carvacrol and linalool) against the castor bean tick, Ixodes ricinus (Acari: Ixodidae).

The castor bean tick, Ixodes ricinus, is a species of medical and veterinary importance. The use of synthetic acaricides for tick control has led to development of resistance, residues in the environment and animal products, and public health concerns. In this regard, plant essential oils and their main constituents represent an appealing alternative strategy to combat ticks. The phenols thymol and carvacrol and the alcohol linalool are monoterpenoids occurring in essential oils of several aromatic and medicinal plants, such as thyme, oregano, savory, lavender and coriander. Recent studies have shown toxicity of these monoterpenoids against selected mosquito vectors and other arthropod pests. However, information on their bioactivity on I. ricinus is not available. On this basis, here we evaluated the ovicidal, larvicidal and repellency effects of these compounds against I. ricinus. Concentrations of 0.25, 0.5, 1, 2 and 5% were sprayed on the egg masses, then hatching rates were noted. Larvicidal assays were conducted on unengorged larvae, following the larval packet technique. The repellency was determined by measuring the vertical migration behavior of ticks in laboratory conditions. Carvacrol and thymol at all concentrations tested led to a significant hatching decrease, showing an efficacy higher than permethrin, whereas linalool did not cause any significant effect. In the larvae treated with carvacrol and thymol (1, 2 and 5%), mortality rates reached 100% after 24h, showing a larvicidal efficacy higher than permethrin, whereas no effect was seen in the larval groups treated with linalool. Carvacrol and thymol at all concentrations tested showed >90% repellency on I. ricinus. Linalool was scarcely effective (50.24% repellency) only at the concentration of 5%. Overall, based on these results, the phenols carvacrol and thymol can be considered as candidate ingredients for the development of novel acaricidal formulations to control the populations of I. ricinus and the spread of related tick-borne diseases.

Acaricide resistance in Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum collected from Haryana and Rajasthan states of India.

Acaricide resistance status of Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum ticks against deltamethrin and diazinon was assessed in Hisar (Haryana) and its adjoining district Churu (Rajasthan) using adult immersion test (AIT) and larval packet test (LPT). The mortality slope, LC50, LC95, 95 % confidence limit and resistance factor of field ticks were determined. Results showed that R. (B.) microplus ticks collected from Tohana (Hisar) were found resistant to both the acaricides while the ticks of Agroha (Hisar) were found to be susceptible using AIT. Similar results were observed by using standard method of LPT. Again, H. anatolicum tick isolates of Tara Nagar (Churu) were found susceptible whereas Churu tick isolates were found to be resistant using AIT for both the acaricides. LPT indicated susceptible status of H. anatolicum ticks collected from Churu as well as Tara Nagar for both the acaricides. The study warrants the need for strategic use of available acaricides to overcome the development of acaricide resistance in ticks.

Acaricidal effect of an isolate from Hoslundia opposita vahl against Amblyomma variegatum (Acari: Ixodidae).

BACKGROUND: Hoslundia opposita Vahl. (Lamiaceae), a common local shrub in Ghana, is traditionally known not only for its pharmacological benefits but also for its insecticidal properties. Its acaricidal property, however, has not been investigated. OBJECTIVE: To test the acaricidal effects of the crude extract and fractions of H. opposita leaves as well as to isolate and characterize the acaricidal principles. MATERIALS AND METHODS: The crude methanolic extract, pet. ether, ethyl acetate and aqueous fractions of the leaves of H. opposita were tested against the larvae of the cattle tick, Amblyomma variegatum, using the Larval Packet Test. A bioassay-guided isolation was carried out to identify the acaricidal principle obtained from the ethyl acetate fraction. RESULTS: The active principle was characterised as ursolic acid, a triterpene previously isolated from the leaves of the same plant. The extract and fractions were less potent than the control, malathion (LC(50) 1.14 × 10(-4) mg/ml). Among the plant samples however the crude methanolic extract exhibited the highest effect against the larvae (LC(50) 5.74 × 10(-2) mg/ml), followed by the ethyl acetate fraction (LC(50) 8.10 × 10(-2) mg/ml). Ursolic acid, pet. ether and aqueous fractions however showed weak acaricidal effects with LC(50) values of 1.13 mg/ml, 8.96 × 10(-1) mg/ml and 1.44 mg/ml, respectively. CONCLUSION: Ursolic acid was not as potent as the crude methanolic extract and the ethyl acetate fraction from which it was isolated. The overall acaricidal effect of H. opposita may have been due to synergy with other principles having acaricidal properties.