Acaricide activity of different extracts from Piper tuberculatum fruits against Rhipicephalus microplus

Rhipicephalus microplus is the main tick that affects cattle. Plant bioactive molecules can be used to control this ectoparasite. The aim of this study was to evaluate the in vitro efficacy of Piper tuberculatum fruit extracts obtained with different solvents on R. microplus larvae and engorged females. Hexane, ethyl ether, ethanolic, and methanolic extracts of P. tuberculatum fruits were evaluated. After extraction, all of the extracts were dried. Adult immersion tests and larval packet tests were performed with five different concentrations of each of the extracts. The hexane extracts of P. tuberculatum showed the highest larvicidal activity against R. microplus (lethal concentration (LC₅₀?=?0.04 mg/mL), followed by the ethyl ether (LC₅₀?=?0.08 mg/mL), ethanolic (LC₅₀?=?2.73 mg/mL), and methanolic (LC₅₀?=?4.49 mg/mL) extracts. The P. tuberculatum fruit extracts were also effective against R. microplus-engorged females. Ethyl acetate extracts showed the highest efficiency (LC₅₀?=?18.4 mg/mL), followed by the methanolic (LC₅₀?=?105.6 mg/mL), ethanolic (LC₅₀?=?140.0 mg/mL), and hexane (LC₅₀?=?297.4 mg/mL) extracts. All of the extracts showed similar chromatographic profiles containing 24 % piperine. The P. tuberculatum fruit extracts contain bioactive compounds with great potential to improve the standard formulations of acaricides for the control of R. microplus.     

In vitro acaricidal activity of Crescentia cujete L. fruit pulp against Rhipicephalus microplus

Cattle tick (Rhipicephalus microplus) is an important constraint on livestock production, particularly in tropical and subtropical areas. Synthetic acaricides are the primary method of control this thick. Resistance of R. microplus to synthetic acaricides has given rise to the need of new scientific investigations on alternative ways to control it. The objective of this work was to conduct a chemical investigation of Crescentia cujete L. fruit pulp and to evaluate the acaricide effect on R. microplus larvae of extracts, fractions, and isolated substances from this species. Crescentia cujete is a Bignoniaceae and it is characteristic of tropical climates. Its most distinctive feature is its fruit, which is spherical and gourd-like, with a hard woody shell and gelatinous pulp. The fruit pulp of C. cujete was extracted with ethanol by maceration yielding an extract and a residue. The extract was partitioned giving an ethyl acetate phase (EAF) that was fractionated by chromatographic procedures yielding cinnamic acid and benzoic acid. The remained botanic material from the ethanol extraction was further and successively extracted with ethyl ether and methanol under reflux. The acaricide effect of all extracts and fractions was evaluated against R. microplus using the larval packet test. The best result was observed for the EAF that caused 100% of mortality when a 10% solution was used and with LC₅₀ of 5.9%. Cinnamic acid was also tested showing 63.0% of mortality with LC₅₀ of 6.6% at the same concentrations and time of the experiment. The data obtained in this study provides information related to the chemical nature and the acaricide activity of the C. cujete epicarp that has not been previously reported. This study shows that the ethyl acetate phase of the ethanol extract of C. cujete fruit pulp is a potential alternative control for R. microplus and its activity is in part associated to cinnamic acid, its major compound.

     

In vitro and in vivo efficacy of Acorus calamus extract against Rhipicephalus (Boophilus) microplus

To develop a environment friendly control measure against cattle tick, Rhipicephalus (Boophilus) microplus, medicinally important plants were identified and extracts were prepared. Twelve 95% ethanolic, thirteen 50% hydroethanolic and nine hot water extracts were prepared and tested against laboratory reared homogenous colony of R. (B.) microplus. Amongst the 34 extracts, 26 extracts showed no mortality within 72 h of application while 12.0 ± 4.9% to 35.0 ± 9.6% mortality of treated ticks was recorded in other extracts. Of the effective extracts, the extract prepared from rhizome of Acorus calamus proved highly efficacious and 100% final mortality within 14 DPT was recorded. The LC85 value of the extract was determined as 11.26. In vivo experiments confirmed the efficacy of the extract up to 42%, and repeat application was required after 7 DPT. The extract was found safe and no reaction was observed when animals were treated with 50% of the concentration, which was five times of the concentration used for in vivo studies. The possibility of using the extract for the control of cattle tick is discussed.     

Acaricidal effect of herbal extracts against cattle tick Rhipicephalus (Boophilus) microplus using in vitro studies

The crude methanolic extract of Datura stramonium, Azadirachta indica, and Calotropis procera leaves, Allium sativum (AS) cloves, and Carica papaya (CP) seeds collected from Banaskanta, Gujarat (India) was tested for its acaricidal properties against Rhipicephalus (Boophilus) microplus. The percent adult mortality within 15 days, reproductive index, percentage inhibition of oviposition, hatching of laid ova, and percentage larval mortality were studied at concentrations of 12.5, 25, 50, and 100 mg/ml. At the highest concentration (100 mg/ml), the adult tick mortality was 66.67, 73.33, 80.00, and 93.33 % for C. procera, D. stramonium, A. sativum, and C. papaya extracts, respectively, and it was statistically significant (P?<?0.001). However, for A. indica, mortality was low and estimated to be 33.33 %. Inhibition of oviposition at the highest concentration of A. indica, C. procera, D. stramonium, A. sativum, and C. papaya extract-treated ticks was 20.73, 71.34, 77.17, 85.83, and 100.00 %, respectively. Inhibition of fecundity of treated groups differed significantly from the control and was concentration dependent. Larvae treated with all the tested concentrations of A. indica, C. procera, D. stramonium, A. sativum, and C. papaya extracts by larval packet test showed significant mortality (P?<?0.001) than that of control tick larvae, and at the highest concentration, it was 55.2, 63.2, 71.8, 69.0, and 82.2 %, respectively. Garlic cloves and papaya seed extract produced complete failure of eclosion of eggs from the treated ticks even at lower concentrations; however, neem, calotropis, and datura was capable of reducing hatchability by 20, 50, and 70 %, respectively. The results pointed that the crude extracts of A. sativum cloves and C. papaya seeds have very good acaricidal properties and could be a potential component of alternative R. (B.) microplus tick control strategy.     

Acaricide and ovicide activities of thymol on engorged females and eggs of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae)

The present work had the objective of evaluating the influence of different concentrations of thymol on the biological parameters of engorged females of Rhipicephalus (Boophilus) microplus and also its ovicide activity on eggs of this tick. In order to carry out the work, four groups were formed, each containing 20 engorged females, which were immersed for 5 min in different concentrations of thymol (1.0%, 1.5%, and 2.0%) and a control group (water + dimethylsulfoxide). The following biological parameters were observed: initial weight (mg); egg mass weight (mg); pre-oviposition, oviposition, and survival period (days); hatching percentage (%HP); egg production (%EPI) and nutritional (%NI) indexes; and the percentage of control (%C). In order to perform the second stage, thymol solutions were sprayed on the egg masses (50 mg). The parameters’ initial weight and pre-oviposition did not present significant differences (p?>?0.05) among the groups. Thymol caused alterations in the parameters egg mass weight, oviposition, survival period, hatching percentage, EPI, and NI, presenting highly significant differences between the treatments and the control group (p?<?0.01). The control efficacy was higher than 95% in all the treatments, reaching 99% in the concentration of 2.0%. In the ovicide test, the hatching percentage was not affected in any of the treatments (p?>?0.05). In the second experiment, when eggs were treated directly, the thymol did not affect significantly this parameter. These results demonstrate that thymol showed deleterious action on most of the analyzed parameters; thus, it is possible to conclude that, in laboratorial conditions, this monoterpene was efficient in the control of engorged females of R. (B.) microplus.     

Acaricidal activity of Cymbopogon winterianus, Vitex negundo and Withania somnifera against synthetic pyrethroid resistant Rhipicephalus (Boophilus) microplus

Detection of resistance levels against cypermethrin and deltamethrin, the most commonly used synthetic pyrethroids (SPs) against Rhipicephalus (Boophilus) microplus collected from Moga, Punjab (India) was carried out using larval packet test. Results indicated the presence of resistance of level I and III against cypermethrin (resistance factors (RF)?=?4.67) and deltamethrin (RF?=?34.2), respectively. Adult immersion test was used to assess the acaricidal activity of aqueous and ethanolic extracts of leaves of Cymbopogon winterianus, Vitex negundo, and Withania somnifera along with roots of V. negundo against the SP resistant engorged females of R. (B.) microplus. The efficacy of various extracts was assessed by estimation of percent adult mortality, reproductive index (RI), percent inhibition of oviposition (%IO), and hatching rate. A concentration dependent increase in tick mortality was recorded which was more marked with various ethanolic extracts, and highest mortality was recorded in ticks treated with ethanolic extract of leaves of C. winterianus. The LC₅₀ values were determined by applying regression equation analysis to the probit transformed data of mortality for various aqueous and ethanolic extracts. Acaricidal property was recorded to be higher in ethanolic extracts, and high activity was found with the ethanolic extract of leaves of C. winterianus with LC₅₀ (95 % CL) values of 0.46 % (0.35–0.59 %), followed by W. somnifera as 5.21 % (4.45–6.09 %) and V. negundo as 7.02 % (4.58–10.74 %). The egg mass weight of the live ticks treated with different concentrations of the various extract was significantly (p?<?0.01) lower than that of control ticks; consequently, the RI and the %IO value of the treated ticks were reduced. Further, complete inhibition of hatching was recorded in eggs laid by ticks treated with ethanolic extracts of leaves of V. negundo and aqueous extracts of leaves of W. somnifera. The results of the current study indicate that extracts of C. winterianus, V. negundo, and W. somnifera can be used for the control of SP resistant ticks.     

In vitro acaricidal efficacy of plant extracts from Brazilian flora and isolated substances against Rhipicephalus microplus (Acari: Ixodidae)

The tick Rhipicephalus microplus causes significant losses in livestock cattle and has developed increasing resistance to the primary acaricides that are used to treat these infections. The objective of this study was to identify new biomolecules or isolated substances showing acaricidal activity from plants. Larval packet tests were conducted to evaluate the effects of 11 species of plants and three isolated substances (betulinic acid, eugenol, and nerolidol) on R. microplus. An adult female immersion test was performed with the substance that showed the highest larvicidal activity, which was evaluated for inhibition of reproduction. Tests using Licania tomentosa, Hymenaea stigonocarpa, Hymenaea courbaril, Stryphnodendron obovatum, Jacaranda cuspidifolia, Jacaranda ulei, Struthanthus polyrhizus, Chrysobalanus icaco, Vernonia phosphorea, Duguetia furfuracea, and Simarouba versicolor extracts as well as the isolated substance betulinic acid indicated lower acaricidal effects on R. microplus larvae. The extract displaying the best larvicidal activity was the ethanolic extract from L. tomentosa at a concentration of 60 %, resulting in a mortality rate of 40.3 %. However, nerolidol and eugenol showed larvicidal activity, which was highest for eugenol. Nerolidol caused a 96.5 % mortality rate in the R. microplus larvae at a high concentration of 30 %, and eugenol caused 100 % mortality at a concentration of 0.3 %. In the adult immersion test, 5 % eugenol was identified as a good biomolecule for controlling R. microplus, as demonstrated by its high acaricidal activity and inhibition of oviposition.     

Nomuraea rileyi as biological control agents of Rhipicephalus microplus tick

Nomuraea rileyi, a fungus pathogenic to insects, has been widely used for biological control of agricultural pests in Brazil. This study investigates the effects of N. rileyi, isolates Nr 138, Nr 151, and Nr 177, to eggs, larvae, and engorged females of Rhipicephalus microplus tick. Specimens were immersed in 1?ml of conidial suspension for 3?min, whereas the control group was immersed in 0.01?% Tween 80 water solution. The isolate Nr 138 controlled 67.37?% of ticks when the highest conidial concentration was used, 10(8) conidia ml(-1). The isolate Nr 177 significantly reduced the percentage of hatch of larvae from eggs treated with 10(8) conidia ml(-1). Conversely, the isolate Nr 151 was not virulent to eggs, larvae, or adults. Variability in virulence was observed among the N. rileyi isolates investigated in the current study-Nr 138 was more virulent to engorged females, while Nr 177 was more virulent to unfed larvae. Although N. rileyi proved to be virulent to several stages of R. microplus, the results obtained in this study indicate that N. rileyi does not appear to be a remarkable biological control agent for R. microplus.     

Acaricidal activity of the hyacinthacine analogues derived from pyrrolizidine alkaloids on Rhipicephalus (Boophilus) microplus

This study reports the effect of six hyacinthacine analogues derived from pyrrolizidine alkaloids on egg hatchability and mortality rates of newly hatched larvae of the cattle tick Rhipicephalus (Boophilus) microplus. All the compounds were toxic to the larvae of the ticks and inhibited the eggs hatchability in the higher concentration tested (5 microg/ml). Even in the lowest concentration (0.625 microg/ml), some effect in the eggs hatchability was observed.     

Spectroscopic evaluation of thymol dissolved by different methods and influence on acaricidal activity against larvae of Rhipicephalus microplus (Acari: Ixodidae)

The acaricidal activity of three thymol formulations was investigated at five concentrations (1.25, 2.5, 5.0, 7.5, and 10.0?mg/ml) on Rhipicephalus microplus larvae, and the behavior of its solubility in these formulations was analyzed. The thymol was dissolved in distilled water plus 1?% dimethylsulfoxide as adjuvant under two heating regimes (water bath in formulation 1 and hot plate in formulation 2) as well as without heating in 50?% ethanol and 50?% water (v/v). The acaricidal activity was assessed by the modified larval packet test, and the solubilization behavior was investigated by ultraviolet–visible spectroscopy, based on the Beer–Lambert law. With formulations 1 and 2, the mortality was greater than 95?% starting at the thymol concentrations of 5.0 and 7.5?mg/ml, respectively, while with formulation 3, this mortality level was reached starting at a concentration of 2.5?mg/ml, showing that the addition of ethanol in the solution enhanced the acaricidal action of thymol. This result was supported by the LC 90 values, which were 3.3, 2.4, and 1.6?mg/ml of thymol for formulations 1, 2, and 3, respectively. This result is related to the better solubility of thymol in the hydroethanolic formulation, since the spectroscopic analysis revealed that the thymol dissolved more completely in this formulation. This fact was evident once the R ² obtained from the linear regression analysis of the relation absorbance × concentration of the formulations 1, 2, and 3 approached the optimal value (R ²?=?1) in the following sequence: 1, 2, and 3 (0.717, 0.901, and 0.968, respectively).     

Repellent activity of eugenol on larvae of Rhipicephalus microplus and Dermacentor nitens (Acari: Ixodidae)

The repellent activity of eugenol was evaluated on Rhipicephalus microplus and Dermacentor nitens larvae in a bioassay based on the vertical migration of these tick larvae during the host-seek phase. Solutions were used at concentrations of 10, 20, 30, 40, and 50 μl/ml and a control only with the solvent ethanol 50 GL. The larvae's reaction of climbing the rods of filter paper was observed each hour for 12 h and once more at 24 h. The repellence was calculated as a percentage through visual estimation, from the tendency of the larvae to climb the rods when the top part was impregnated with the eugenol solutions. For R. microplus larvae, the four highest concentrations produced repellence levels were greater than 80 % for up to 9 h. The repellence rates after 10 h for the concentration of 10 μl/ml was lower than 2 % (p?>?0.05); and after 24 h, the values observed for the concentrations of 40 and 50 μl/ml were 77 and 80 % (p?<?0.05). For the D. nitens larvae, the repellence was greater than 80 % for up to 5 h at the concentrations of 40 and 50 μl/ml; and after 9 h, the repellence fell for all treated groups were significantly different than the control were found only at the three highest concentrations. Therefore, eugenol has repellent activity on the larval stage of these two tick species, with the R. microplus larvae being more sensitive.     

Acaricide rotation strategy for managing resistance in the tick Rhipicephalus (Boophilus) microplus (Acarina: Ixodidae): laboratory experiment with a field strain from Costa Rica

During the past two decades, resistance to pyrethroids within the cattle tick genus Boophilus has caused tick control problems in various tropical countries, mainly in Latin America, southern Africa, Australia, and New Caledonia. A Rhipicephalus (Boophilus) microplus (Canestrini) strain from Costa Rica, exhibiting resistance to the pyrethroid deltamethrin but only a very low resistance to organophosphates (OP) was kept under selection pressure for 9 to 11 generations by using deltamethrin or coumaphos (OP), either exclusively or in rotation. The objective of this acaricide rotation was to examine the possibility of delaying or reducing the full emergence of pyrethroid resistance. In the substrain selected with deltamethrin at the LD50 concentration, resistance to deltamethrin was measured after five generations (resistance factor [RF] = 9.2) and very high resistance after 11 generations (RF = 756), compared with the starting field strain from Costa Rica. In the substrain selected with deltamethrin then coumaphos in rotation, resistance to deltamethrin was variable from one generation to the next (RF = 1-4.2), but no high, stable resistance developed. After 10 generations of rotation, the deltamethrin RF was 1.6. In the substrains selected continuously with coumaphos or coumaphos and deltamethrin in rotation, no consistent change in resistance to coumaphos was observed. Rotation of deltamethrin with coumaphos seems to delay the development of strong resistance to deltamethrin in a population that had initially a low level of deltamethrin resistance.     

Seasonal analysis and acaricidal activity of the thymol-type essential oil of Ocimum gratissimum and its major constituents against Rhipicephalus microplus (Acari: Ixodidae)

Parasitology Research - The tick Rhipicephalus microplus affects cattle health, with production loss in tropical and subtropical regions. Moreover, the use of commercial acaricides has been reduced...     

Efficacy of spinosad against acaricide-resistant and -susceptible Rhipicephalus (Boophilus) microplus and acaricide-susceptible Amblyomma americanum and Dermacentor variabilis

Various acaricide-resistant strains of Rhipicephalus (Boophilus) microplus, representative of the major resistance mechanisms found in Mexico and Brazil, were exposed to spinosad using the Food and Agriculture Organization of the United Nations larval packet test and adult immersion test (AIT). Larvae of all strains tested were found to be susceptible to spinosad. Conversely, spinosad did not show toxic activity toward engorged females used in the AIT. In vitro tests against larvae, nymphs, and adults of acaricide-susceptible Amblyomma americanum and Dermacentor variabilis revealed differences in activity that were dependent on tick species and parasitic life stage. Spinosad seemed to be a viable alternative to current acaricides available for tick control in the species tested. The larval packet test should be used for future monitoring of resistance, as the AIT did not provide useful information with this chemical. The potential benefit for the use of spinosad in integrated pest management or eradication programs is discussed.     

ABC transporters as a multidrug detoxification mechanism in Rhipicephalus (Boophilus) microplus

ATP-binding cassette (ABC) transporters are responsible for pumping drugs across membranes and are an important drug detoxification mechanism. Since ABC transporters act on a wide spectrum of chemical compounds, they have been associated with multidrug resistance phenotype in various parasites and cancer cells. Here, we document the presence of a Rhipicephalus (Boophilus) microplus tick population (Jaguar) resistant to four acaricide classes (organophosphates (OP), synthetic pyrethroids (SP), amitraz and macrocyclic lactones (ML)) and reveal that the cattle tick has a multidrug detoxification mechanism based on ABC transporter proteins. Acaricide toxicity was assessed using the larval packet test (LPT), and mortality data were subjected to probit analysis using a susceptible strain (POA) as reference. Larvae were pre-exposed to sub-lethal doses of the ABC-transporter inhibitors, cyclosporin A (CsA) and MK571, and subsequently treated with ivermectin, abamectin, moxidectin, chlorpyriphos, cypermethrin, or amitraz in LPT. Results show that lethal concentrations 50?% (LC₅₀) of ivermectin, abamectin, moxidectin (MLs), and chlorpyriphos (OP) were significantly reduced in larvae exposed to CsA and MK571 inhibitors in the Jaguar resistant population, but LC₅₀ did not change in POA susceptible strain larvae. LC₅₀ of cypermetrin (SP) and amitraz remained unchanged in inhibitor-exposed larvae, compared to larvae from Jaguar and POA strains not exposed to inhibitor. These results suggest that ABC transporter proteins can protect ticks against a wide range of acaricides and have an important implication in drug resistance development as a multidrug detoxification mechanism.     

Acaricidal activity of extract of Artemisia absinthium against Rhipicephalus sanguineus of dogs

The objective of the study was to evaluate the in vitro efficacy of different concentrations of chloroform extract obtained from the aerial parts of Artemisia absinthium in comparison to amitraz on adults, eggs and larvae of the dog tick Rhipicephalus sanguineus using the adult immersion test (AIT), egg hatchability test (EHT) and larval packet test (LPT), respectively. Five concentrations of the extract (1.25, 2.5, 5, 10 and 20 %) with three replications for each concentration were used in all the bioassays. A control group was established (water + dimethylsulphoxide) together with a positive control group (amitraz), with three repetitions each. In AIT, the mortality rates were 0.0, 13.3, 16.7, 33.3 and 93.3 % in concentrations of 1.25, 2.5, 5, 10 and 20 %, respectively, and the variation was significant (p?=?0.0151). The LC₅₀ (CI) and LC₉₅ (CI) values were calculated as 8.793 % (8.217–9.408) and 34.59 % (29.71–40.26), respectively. The egg production was reduced by 6.6, 6.6, 18.3, 42.5 and 85.1 % in the concentrations of 1.25, 2.5, 5, 10 and 20 %, respectively, and it was statistically significant (p?=?0.0274). In EHT, hatching was completely inhibited at 5, 10 and 20 % displaying 100 % ovicidal action while at the concentrations of 1.25 and 2.5 %, the inhibition rates were 20 and 60 %, respectively. In LPT, the extract caused 100 % mortality of larvae in the concentrations of 5, 10 and 20 % after 24 h while at the concentrations of 1.25 and 2.5 %, the mortality rates were 54.3 and 96.7 %, respectively. The LC₅₀ (CI) and LC₉₅ (CI) values were determined to be 1.11 % (1.099–1.121) and 2.37 % (2.328–2.423), respectively. The results show that the extract of A. absinthium has acaricidal properties and could be useful in controlling R. sanguineus which is an efficient vector of pathogens both in dogs and humans.     

Differential immunoproteomics enables identification of Metarhizium anisopliae proteins related to Rhipicephalus microplus infection

Differential immunoproteomics was applied to identify proteins secreted by Metarhizium anisopliae induced by the Rhipicephalus microplus cuticle. In addition, IgG anti-spore surface proteins were used for searching for proteins possibly involved in early stages of fungus versus tick infection. LC-MS/MS of differentially secreted proteins led to the identification of proteases (carboxypeptidase and Pr1A), chitinase, carboxylic acid transport and proline-rich protein. Differential immunoproteomics strategy facilitated the detection and the identification of new proteins related to M. anisopliae host–pathogen interaction and could be used in further works to identify novel proteins related to other microbial infection systems.