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.

Histopathological study of ovaries of Rhipicephalus sanguineus (Acari: Ixodidae) exposed to different thymol concentrations.

Thymol is a monoterpene with proven acaricide action for several tick species. In addition to killing these ectoparasites, thymol can also reduce oviposition and egg hatch rate. However, the effects of thymol on the morphophysiology of tick ovaries are still unknown. Thus, the aim of this study was to evaluate the morphophysiological changes caused by this active principle in ovaries of Rhipicephalus sanguineus after a 6-day feeding period, through the application of morphohistochemical techniques. After the feeding period, a total of 50 females were divided into five groups and immersed in the following solutions: (I) distilled water (control), (II) 30% ethanol (control), (III) 1.25 mg/mL thymol, (IV) 2.5 mg/mL thymol, and (V) 5.0 mg/mL thymol. The experimental groups were kept in a climatic chamber (27 ± 1 °C; RH 80 ± 10%) for 5 days. After this period, morphological (hematoxylin/eosin) and histochemical (von Kossa) techniques were applied after remotion of the ovaries. The morphological results revealed large vacuoles in germ cells at different developmental stages and invaginations that represent deformations in the chorionic membrane. From the results obtained in this study, it was concluded that thymol interfered with the development of oocytes, which showed degeneration signs. The treatment containing 5.0 mg/mL thymol affected more accentuately the morphological development. Moreover, thymol also altered the calcium content of yolk granules, which generally showed an intense staining for this element.

Entomopathogenic nematodes in insect cadaver formulations for the control of Rhipicephalus microplus (Acari: Ixodidae).

This study evaluated the efficacy of four entomopathogenic nematode (EPN) strains in insect cadaver formulations against Rhipicephalus microplus and compared the efficacy of the most virulent EPNs applied in cadavers of Galleria mellonella and Tenebrio molitor. In the first experiment, infected G. mellonela larvae were used as the source of EPNs. Engorged females of R. microplus were placed in pots filled with soil and different numbers of G. mellonella larvae infected with one of four species of nematodes. All treatments with EPNs of the genus Heterorhabditis caused significant reduction (p<0.05) in the egg mass weight and hatching percentage of larvae. The EPNs of the genus Steinernema, except for the group exposed to Steinernema carpocapsae ALL, whose source nematodes included six larvae of G. mellonella, caused a significant reduction (p<0.05) in the egg mass weight produced per female. Steinernema feltiae SN applied with two, four, and six cadavers and S. carpocapsae ALL with two cadavers caused a reduction in hatching percentage of larvae of R. microplus (p<0.05). The percentage of control was above 95% in all groups treated with Heterorhabditis bacteriophora HP88 and Heterorhabditis indica LPP1 and in the treatment with four larvae infected with S. feltiae SN. The second experiment followed the same methodology, using G. mellonella and T. molitor larvae infected by the two most virulent EPNs. H. bacteriophora HP88 and H. indica LPP1 in different formulations caused reduction in the egg mass weight and hatching percentage of larvae. The percentage of control were 82.4 and 84.9% for H. bacteriophora HP88 and H. indica LPP1, respectively, formulated in T. molitor, and reaching 99.9% in groups formulated with G. mellonella. The EPNs tested in insect cadaver formulation showed pathogenicity to engorged females of R. microplus and EPNs of the genus Heterorhabditis formulated in G. mellonella larvae were more effective.

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.

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.