Daylight photodynamic inactivation of cattle tick Rhipicephalus microplus by porphyrins: An alternative for the ectoparasite control.

The bovine tick Rhipicephalus microplus, a primary ectoparasite of veterinary concern, contributes significantly to disease transmission and reduced cattle productivity, resulting in substantial economic losses. The overuse of chemical acaricides has led to the emergence of resistant strains, posing a considerable challenge to veterinary medicine. Consequently, the development of alternative parasite control methods is essential to ensure livestock quality and enhance food safety worldwide. Our study introduces an innovative approach to photodynamic inactivation (PDI) of the bovine tick, harnessing natural daylight for a potential field application. Reproductive parameters (female and egg mass, egg production index, and larval hatch) were evaluated in engorged female ticks under photodynamic action using the hematoporphyrin (HP) and tetra-cationic porphyrins free-base meso-tetra-ruthenated (4-pyridyl) (RuTPyP) and its zinc(II) complex (ZnRuTPyP) as photosensitizers (PS). The results showed that there was no significant difference between the groups treated with tetra­ruthenium porphyrins and the control group. However, HP exhibits a control percentage of 97.9% at a concentration of 2.5 µmol.L-1, aligning with the expected control rates achieved by conventional chemical acaricides. Photophysical and physicochemical parameters such as the number of singlet oxygen produced and lipophilicity were discussed for each PS and related to tick control percentages. Furthermore, the interaction between HP and chitin, an important macromolecule presents in the tick's cuticle, considered as the primary target tick structure during PDI was observed by the absorption and fluorescence emission spectroscopic techniques. Therefore, the results presented here extend the potential for controlling R. microplus through photodynamic inactivation while utilizing sunlight as a source of natural irradiation.

Two protocols using fluralaner for Rhipicephalus microplus strategic control on taurine cattle in a tropical region.

BACKGROUND: The present study aimed to evaluate the effects of different treatment strategies using a new commercial formulation containing pour-on fluralaner on Rhipicephalus microplus infestation in cattle and in pastures in a tropical climate region where up to five generations of this tick species can occur per year. METHODS: Forty-five naturally infested cattle were divided into three experimental groups: T01, treated with fluralaner (2.5 mg/kg) pour-on every 42 days; T02, the cattle received the first treatment with fluralaner on Day 0 but the next treatment involved a weekly visual evaluation; T03, control, received palliative treatment with a spray formulation when the group mean was ≥ 30 ticks. Counts of female R. microplus were performed weekly until day 343, and larval counts on pasture were performed on Days 0, 30, and 60 and every 30 days until Day 330. RESULTS: Using fluralaner, six applications were performed in Group T01, and four were performed in Group T02. In the control group (T03), it was necessary to perform eight palliative acaricide treatments with the spray formulation. The animals in T01 and T02 showed lower mean tick counts (p ≤ 0.05) than the control group (T03) on 28 and 27 of the 49 evaluated dates, respectively. In the paddock where the animals were kept as controls, the R. microplus larvae counts increased to 1458. In the paddocks where the animals were treated with fluralaner, the number was ≤ 19 per paddock during the study. CONCLUSIONS: The different strategic treatment protocols performed with pour-on fluralaner (2.5 mg/kg) over a year in taurine cattle in a tropical region with a history of up to five annual generations of cattle ticks were effective, maintaining levels of R. microplus infestations in animals and in pastures close to zero in most of the study. Depending on the retreatment criterion adopted, the number of applications per year may be lower, resulting in a reduction in the mean cost of acaricide treatment per year and lower exposure of R. microplus populations to the active ingredient, resulting in lower resistance and selection pressure.

Influence of the acaricide emulsion pH on the effectiveness of spray products to control the cattle tick: laboratory and field investigations.

The current work evaluated the efficacy of 10 commercial acaricides in different pHs (4.5, 5.5, and 6.5) in laboratory (adult immersion tests (AIT), pH evaluation over time) and field assays (tick counts and efficacy). In the AIT (n=70), higher efficacies were obtained when the acaricide emulsion had a more acidic pH (4.5), mainly for two combinations of pyrethroids + organophosphate (acaricide 3 and acaricide 9). For amidine, a higher pH (6.5) showed a higher efficacy. Over time, there was a trend in the pH of these emulsions increasing. When the efficacy of chlorpyrifos + cypermethrin + piperonyl butoxide (acaricide 3) at different pHs was evaluated over time (0, 6, 12, and 24h) by AIT, the less acidic pH (6.5) showed a strongly variation in the acaricide efficacy range. The mean pH of the water samples from different regions of Brazil was 6.5. In the field, the association of pyrethroid + organophosphates (acaricide 9) with pH of 4.5 and 5.5 were more effective in tick control than the emulsion prepared with this same spray formulation at pH 6.5. The pH of the acaricide emulsions is an important point of attention and is recommended that the veterinary industry start to develop/share information regarding how the pH can affect the acaricide efficacy.

Acaricidal activity of essential oils of Cinnamomum zeylanicum and Eremanthus erythropappus, major compounds and cinnamyl acetate in Rhipicephalus microplus.

This study aimed to chemically characterize the essential oils (EOs) of Cinnamomum zeylanicum (cinnamon) and Eremanthus erythropappus (candeia) and evaluate their acaricidal activity, together with that of their major compounds and cinnamyl acetate derivative, against Rhipicephalus microplus. Essential oil compounds were identified through gas chromatography. The larval packet test (LPT) at concentrations ranging from 0.31 to 10.0 mg/mL and the adult immersion test (AIT) at concentrations between 2.5 and 60.0 mg/mL were performed. (E)-cinnamaldehyde and α-bisabolol were the major compounds in cinnamon (86.93%) and candeia (78.41%) EOs, respectively. In the LPT, the EOs of cinnamon and candeia and the compounds (E)-cinnamaldehyde, α-bisabolol and cinnamyl acetate resulted in 100% mortality at concentrations of 2.5, 2.5, 5.0, 10.0 and 10.0 mg/mL respectively. In the AIT, percentage control values > 95% were observed for cinnamon and candeia EOs, (E)-cinnamaldehyde and α-bisabolol at the concentrations of 5.0, 60.0, 20.0, and 20.0 mg/mL, respectively, whereas cinnamyl acetate showed low activity. We conclude that EOs and their compounds showed high acaricidal activity, whereas the acetylated derivative of (E)-cinnamaldehyde presented less acaricidal activity on R. microplus engorged females.

Acaricidal activity of essential oils of Cinnamomum zeylanicum and Eremanthus erythropappus, major compounds and cinnamyl acetate in Rhipicephalus microplus / Atividade acaricida dos óleos essenciais de Cinnamomum zeylanicum e Eremanthus erythropappus, compostos majoritários e acetato de cinamila sobre Rhipicephalus microplus

Abstract This study aimed to chemically characterize the essential oils (EOs) of Cinnamomum zeylanicum (cinnamon) and Eremanthus erythropappus (candeia) and evaluate their acaricidal activity, together with that of their major compounds and cinnamyl acetate derivative, against Rhipicephalus microplus. Essential oil compounds were identified through gas chromatography. The larval packet test (LPT) at concentrations ranging from 0.31 to 10.0 mg/mL and the adult immersion test (AIT) at concentrations between 2.5 and 60.0 mg/mL were performed. (E)-cinnamaldehyde and α-bisabolol were the major compounds in cinnamon (86.93%) and candeia (78.41%) EOs, respectively. In the LPT, the EOs of cinnamon and candeia and the compounds (E)-cinnamaldehyde, α-bisabolol and cinnamyl acetate resulted in 100% mortality at concentrations of 2.5, 2.5, 5.0, 10.0 and 10.0 mg/mL respectively. In the AIT, percentage control values > 95% were observed for cinnamon and candeia EOs, (E)-cinnamaldehyde and α-bisabolol at the concentrations of 5.0, 60.0, 20.0, and 20.0 mg/mL, respectively, whereas cinnamyl acetate showed low activity. We conclude that EOs and their compounds showed high acaricidal activity, whereas the acetylated derivative of (E)-cinnamaldehyde presented less acaricidal activity on R. microplus engorged females.

Resumo Este estudo teve como objetivo caracterizar quimicamente os óleos essenciais (OE) de Cinnamomum zeylanicum (canela) e Eremanthus erythropappus (candeia) e avaliar sua atividade acaricida, juntamente com a de seus principais compostos e do derivado de acetato de cinamila, sobre Rhipicephalus microplus. Os compostos do óleo essencial foram identificados por cromatografia gasosa. Foram realizados o Teste de Pacote de Larvas (TPL), em concentrações variando de 0,31 a 10,0 mg/mL, e o Teste de Imersão de Adultos (TIA), em concentrações entre 2,5 e 60,0 mg/mL. (E)-cinnamaldeído e α-bisabolol foram os principais compostos nos OE da canela (86,93%) e da candeia (78,41%), respectivamente. No TPL, os OEs de canela e candeia, e os compostos (E)-cinnamaldeído, α-bisabolol e acetato de cinamila resultaram em 100% de mortalidade nas concentrações de 2,5, 2,5, 5,0, 10,0 e 10,0 mg/mL, respectivamente. No TIA, valores percentuais de controle >95% foram observados para OE de canela e candeia, (E)-cinnamaldeído e α-bisabolol nas concentrações de 5,0, 60,0, 20,0 e 20,0 mg/mL, respectivamente, enquanto o acetato de cinamila apresentou baixa atividade. Conclui-se que os OEs e seus compostos apresentaram alta atividade acaricida, enquanto o derivado acetilado do (E)-cinnamaldeído apresentou menor atividade acaricida em fêmeas ingurgitadas de R. microplus.

Activity of the extract of Acmella oleracea on immature stages of Amblyomma sculptum (Acari: Ixodidae).

This study evaluated the acaricidal activity of the methanol extract of Acmella oleracea with 0.187% of spilanthol against immature stages of Amblyomma sculptum. The packet test was used to evaluate the extract's activity on unengorged larvae and nymphs, testing concentrations of 0.4 to 50 mg/mL for larvae and 12.5 to 200.0 mg/mL for nymphs. For the engorged stages, the immersion test was used, at concentrations of 0.4 to 50 mg/mL for larvae and 12.5 to 200.0 mg/mL for nymphs. The methanol extract caused 100% mortality of the unengorged larvae and nymphs starting at concentrations of 12.5 and 200.0 mg/mL, respectively. For engorged larvae and nymphs, the mortality was 100% starting from concentrations of 12.5 and 150.0 mg/mL, respectively. The LC50 for unengorged larvae was 3.2 mg/mL, while for engorged larvae it was 6.6 mg/mL. For unengorged nymphs, the LC50 was 38.5 mg/mL, but it was not possible to calculate the corresponding value for engorged nymphs because the data did not fit the probit model. These results demonstrate that the methanol extract of A. oleracea has acaricidal activity against different immature stages of A. sculptum.

Acaricidal activity of methanol extract of Acmella oleracea L. (Asteraceae) and spilanthol on Rhipicephalus microplus (Acari: Ixodidae) and Dermacentor nitens (Acari: Ixodidae).

We evaluated the acaricidal activity of Acmella oleracea methanol extract and spilanthol on Rhipicephalus microplus and Dermacentor nitens. The extract was made through maceration with methanol. From this extract, a dichloromethane fraction with 99% spilanthol was obtained and tested on R. microplus larvae and engorged females and D. nitens larvae. For evaluation against larvae, the modified larval packet test was used, and both the methanol extract and dichloromethane fraction were tested at concentrations of 0.2-50mg/mL. The modified larval packet test was also used in the lethal time (LT) test, with the methanol extract at a concentration of 12.5mg/mL and the percentage mortality was assessed after 15, 30, 45, 60, 75, 90, 120min and 24h. The 50% lethal time calculation (LT50) was performed in this test. The engorged female test was performed with R. microplus only, at concentrations of 25-200mg/mL for methanol extract and 2.5-20.0mg/mL for spilanthol. The methanol extract caused 100% mortality of the R. microplus and D. nitens larvae at concentrations of 3.1 and 12.5mg/mL, respectively. Spilanthol resulted in 100% mortality of R. microplus larvae at concentration of 1.6mg/mL and of D. nitens at 12.5mg/mL. In the lethal time assay using the methanol extract, the mortality rate was 100% for R. microplus and D. nitens larvae after 120min and 24h, with LT50 values of 38 and 57min, respectively. In the test of females, the egg mass weight and the hatching percentage of the groups treated with concentrations equal to or higher than 50.0mg/mL of methanol extract were significantly reduced (p<0.05), while for spilanthol, the reduction of the egg mass weight and hatching percentage occurred from concentrations of 10.0mg/mL and 2.5mg/mL, respectively. Females treated with 200.0mg/mL of extract died before starting oviposition, resulting in 100% effectiveness, while the best efficacy for spilanthol was 92.9% at a concentration of 20.0mg/mL. Thus we conclude that the methanol extract of A. oleracea and spilanthol have acaricidal activity against R. microplus and D. nitens.

Lab-on-a-chip and SDS-PAGE analysis of hemolymph protein profile from Rhipicephalus microplus (Acari: Ixodidae) infected with entomopathogenic nematode and fungus.

In the present study, lab-on-a-chip electrophoresis (LoaC) was suggested as an alternative method to the conventional polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) to analyze raw cell-free tick hemolymph. Rhipicephalus microplus females were exposed to the entomopathogenic fungus Metarhizium anisopliae senso latu IBCB 116 strain and/or to the entomopathogenic nematode Heterorhabditis indica LPP1 strain. Hemolymph from not exposed or exposed ticks was collected 16 and 24 h after exposure and analyze by SDS-PAGE or LoaC. SDS-PAGE yielded 15 bands and LoaC electrophoresis 17 bands. Despite the differences in the number of bands, when the hemolymph protein profiles of exposed or unexposed ticks were compared in the same method, no suppressing or additional bands were detected among the treatments regardless the method (i.e., SDS-PAGE or chip electrophoresis using the Protein 230 Kit®). The potential of LoaC electrophoresis to detect protein bands from tick hemolymph was considered more efficient in comparison to the detection obtained using the traditional SDS-PAGE method, especially when it comes to protein subunits heavier than 100 KDa. LoaC electrophoresis provided a very good reproducibility, and is much faster than the conventional SDS-PAGE method, which requires several hours for one analysis. Despite both methods can be used to analyze tick hemolymph composition, LoaC was considered more suitable for cell-free hemolymph protein separation and detection. LoaC hemolymph band percent data reported changes in key proteins (i.e., HeLp and vitellogenin) exceptionally important for tick embryogenesis. This study reported, for the first time, tick hemolymph protein profile using LoaC.

Evaluation of the combined effect of thymol, carvacrol and (E)-cinnamaldehyde on Amblyomma sculptum (Acari: Ixodidae) and Dermacentor nitens (Acari: Ixodidae) larvae.

This study aimed at assessing the combined effect of thymol, carvacrol and (E)-cinnamaldehyde on Amblyomma sculptum and Dermacentor nitens larvae. The effects resulting from treatments were evaluated by means of the modified larval packet test. In order to determine the LC50, components of essential oils, the monoterpenes thymol, carvacrol and phenylpropanoid (E)-cinnamaldehyde were individually tested at different concentrations. After determining the LC50, each essential oil component was separately evaluated and then combined with another substance at a 1:1 proportion at the LC50 concentration and at 1/2 and 1/4 of the LC50. For A. sculptum, the lowest LC50 value was obtained for (E)-cinnamaldehyde (1.40 mg/ml), followed by thymol (2.04 mg/ml) and carvacrol (3.49 mg/ml). The same order of effectiveness was observed for D. nitens, with values of 1.68, 2.17 and 3.33 mg/ml, respectively. In the evaluation of component associations of essential oils against A. sculptum larvae, only the combinations between carvacrol and thymol (LC50) and carvacrol and (E)-cinnamaldehyde (1/4 LC50) presented a moderate synergetic effect. In turn, for D. nitens larvae, the combinations between thymol and carvacrol (LC50 and 1/2 LC50) presented a synergetic effect, while the others presented an additive or antagonistic effect. Therefore, it can be concluded that the combination of thymol and carvacrol (LC50) has a moderate synergetic effect against A. sculptum larvae, while thymol, combined with carvacrol (LC50 and 1/2 LC50), has a synergetic effect against D. nitens larvae.

Acaricidal activity of thymol against larvae of Rhipicephalus microplus (Acari: Ixodidae) under semi-natural conditions.

This is the first study to investigate the activity of thymol on Rhipicephalus microplus larvae under semi-natural conditions. For this purpose, tests were conducted in pots with Brachiaria decumbens seedlings containing cattle tick larvae. Thymol, diluted in ethanol 50° GL, was tested at concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 mg/mL, along with the control group treated with the solvent alone. Each treatment was composed of five pots (1 pot = a repetition). The experiment was performed in three steps. On the first day, the larvae were applied at the base of the signalgrass. Twenty-four hours later, approximately 25 mL of the solution was applied with thymol on the top of the vegetation in each pot. The survival of the larvae was measured 24 h after application of the solutions. Each pot was analyzed individually, and the grass fillets contained larvae were cut with scissors, placed in Petri dishes, and taken to the laboratory to count the number of living larvae. At the highest concentrations (10, 15, and 20 mg /mL), the number of live larvae declined by more than 95 % in relation to the control group. The lethal concentration 50 % (LC50) and LC90 values were 3.45 and 9.25 mg/ml, respectively. The application of thymol in semi-natural conditions starting concentration of 10 mg/mL significantly reduced the number of living R. microplus larvae.

Entomopathogenic nematodes associated with essential oil of Lippia sidoides for control of Rhipicephalus microplus (Acari: Ixodidae).

In vitro effect of the association of the entomopathogenic nematodes Heterorhabditis bacteriophora HP88 and Heterorhabditis indica LPP1 with the essential oil of Lippia sidoides on engorged females of Rhipicephalus microplus was assessed. Engorged females with homogeneous weights (p >0.05) were divided into six groups of ten ticks each (each female = an experimental unit). In the treated groups, the ticks were exposed to the oil (40 µl/ml=4%) and nematodes (300 entomopathogenic nematodes (EPNs/tick) separately and also to the nematodes together with the oil, while in the control group, the females were immersed in Tween 80 (3%). All the treatments caused a significant reduction in the egg mass weight (p <0.05). The hatching percentage of larvae was reduced (p <0.05) in the groups treated with H. bacteriophora HP88, H. indica LPP1, and H. bacteriophora HP88 associated with the oil, but in the group treated only with the oil, the hatching of percentage of larvae rate was similar to that of the control group (p >0.05). The percentage of control in the group exposed only to the oil was 50.7%, while in the other treated groups, the percentage of control was greater than 95 %, reaching 100 %in the group treated with H. indica LPP1 associated with the oil. The results of the L. sidoides essential oil in the concentrations tested in association with both H. bacteriophora HP88 and H. indica LPP1 indicate that these combinations can be used to control R. microplus.

Acaricidal activity of essential oil from Lippia sidoides on unengorged larvae and nymphs of Rhipicephalus sanguineus (Acari: Ixodidae) and Amblyomma cajennense (Acari: Ixodidae).

The aims of this work were to identify the compounds and to investigate the acaricidal activity of the essential oil of Lippia sidoides for unengorged larvae and nymphs of Rhipicephalus sanguineus and Amblyomma cajennense. The oil was analyzed by gas chromatography and gas chromatography/mass spectrometry. In total, 22 compounds comprising 98.5% of the total peak area were identified. The major constituent of the essential oil was thymol (69.9%). The acaricidal activity against larvae and nymphs was assessed using a modified larval packet test. In all experiments, oils were tested at concentrations of 2.35, 4.70, 9.40 14.10 and 18.80 mg/mL. The mortalities of larvae and nymphs of R. sanguineus were 20.6, 47.8, 73.6, 99.5 and 99.0% and 12.0, 50.0, 76.3, 96.0 and 96.1%, respectively. For larvae and nymphs of A. cajennense the rates of mortality were 41.9, 63.3, 77.8, 82.5 and 100.0% and 0.0, 32.8, 64.8, 71.1 and 94.0%, respectively. The LC 90 values of the L. sidoides oil were 11.56 and 12.97 mg/mL for larvae and nymphs of R. sanguineus and 15.70 and 18.52 mg/mL for larvae and nymphs of A. cajennense, respectively. The essential oil from L. sidoides has acaricidal activity on unengorged larvae and nymphs of R. sanguineus and A. cajennense.

Determination of the susceptibility of unengorged larvae and engorged females of Rhipicephalus microplus (Acari: Ixodidae) to different methods of dissolving thymol.

The present study evaluated efficacy of thymol against engorged females and unengorged larvae of Rhipicephalus microplus when diluted in 1 % dimethyl sulfoxide (DMSO) (formulation 1) and diluted in hydro-ethanol solutions of 50 % (formulation 2), 40 % (formulation 3), and 30 % (formulation 4). The immersion test was used on the engorged females for all dilution methods, while the larval packet method was applied to the larvae to test formulations 3 and 4 since the other two had already been tested before. Two control groups were established in both experiments; in one, ticks were treated with 1 % DMSO, and in the other, with 50 % ethanol, with ten repetitions for each treatment. The groups were maintained at 27±1 °C and RH>80 ± 10 %. For engorged females, the parameters weight before oviposition, egg mass weight, hatching percentage of larvae, and percentage of control were evaluated, while for larvae, percentage of mortality was evaluated. The egg mass weight of all treatments differed significantly (p<0.05) from the control groups; however, the reduction in egg mass weight was more accentuated (p<0.05) in the treatments with formulations 1, 3, and 4. The values of hatching percentage of the group treated with formulation 2 and control group were statistically similar (p>0.05). The group treated with formulation 2 attained the lowest percentage of control (66 %). In the other treatments, the control percentages were greater than 99 %. In larval test, the mortality was 100 % in all the treatments, and there was no mortality in the control groups.

Association between entomopathogenic nematodes and fungi for control of Rhipicephalus microplus (Acari: Ixodidae).

The aim of the study was to assess the effect of the association of entomopathogenic nematodes and fungi on Rhipicephalus microplus. The nematodes used were Heterorhabditis bacteriophora HP88 and Heterorhabditis indica LPP1 and the fungi were Metarhizium anisopliae IBCB 116 and Beauveria bassiana ESALQ 986. In the groups treated with the fungi, the females were immersed for 3 min in a conidial suspension, while in the groups treated with the nematodes, the ticks were exposed to infective juveniles. To evaluate the interaction between entomopathogens, the females were first immersed in a conidial suspension and then exposed to the nematodes. The egg mass weight and hatching percentage values of the groups treated with M. anisopliae IBCB 116 and B. bassiana ESALQ 986 in the two experiments were statistically similar (p > 0.05) to the values of the control group. In the groups treated only with nematodes, there was a significant reduction (p < 0.05) in the egg mass weight, a fact also observed for the hatching percentage of the group treated with H. indica LPP1. In all the groups treated with nematodes in association with fungi, there was a significant reduction (p < 0.05) in the egg mass weight and hatching percentage. The percentage of control of the groups treated with fungi alone varied from 31 to 55%. In the groups treated with nematodes associated or not with fungi, the control percentage was always greater than 90% and reached 100% in the group treated with H. bacteriophora HP88 associated with the fungus M. anisopliae IBCB 116.

Investigation of activity of monoterpenes and phenylpropanoids against immature stages of Amblyomma cajennense and Rhipicephalus sanguineus (Acari: Ixodidae).

The objective of this study was to assess the acaricidal activity of carvacrol, thymol, eugenol, and (E)-cinnamaldehyde on unengorged larvae and nymphs of Amblyomma cajennense and Rhipicephalus sanguineus, using the modified larval packet test. Carvacrol, eugenol, and (E)-cinnamaldehyde were tested at concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 µl/ml, while thymol was tested at concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 mg/ml, in all cases with 10 repetitions per treatment. For the A. cajennense larvae, mortality rates caused by carvacrol, thymol, eugenol, and (E)-cinnamaldehyde at the lowest concentration were 45.0, 62.7, 10.2, and 81.6%, respectively, reached 100% at the concentration of 5.0 µl/ml for carvacrol and (E)-cinnamaldehyde and 5.0 mg/ml for thymol, while this mortality was observed at 15.0 µl/ml for eugenol. For the nymphs of this species, carvacrol and thymol caused 100% mortality starting at a concentration of 5.0 µl/ml and 10.0 mg/ml, respectively, while eugenol caused 100% mortality at 20.0 µl/ml and the mortality caused by (E)-cinnamaldehyde did not exceed 64%. In the tests with R. sanguineus larvae, the lowest concentration of carvacrol and (E)-cinnamaldehyde resulted in 100% mortality, while this percentage was observed starting at 10.0 µl/ml for eugenol. For nymphs, carvacrol and thymol at the smallest concentration caused 100% lethality, unlike the results for eugenol and (E)-cinnamaldehyde, where 100% mortality was only observed starting at the concentration of 10.0 µl/ml. The results obtained indicate that the tested substances have acaricidal activity on unengorged larvae and nymphs of A. cajennense and R. sanguineus.

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.

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).