Do Commercial Insect Repellents Provide Protection against the Tick Amblyomma sculptum (Acari: Ixodidae)?

Amblyomma sculptum is a species of public health interest because it is associated with the transmission of the bacteria that causes Brazilian Spotted Fever (BSF). The use of repellents on humans is a prophylactic measure widely used to provide protection against a series of arthropod vectors, including mosquitoes and ticks. However, in Brazil, the effectiveness of commercial repellents against A. sculptum is little known. Therefore, it is necessary to carry out specific studies to evaluate the repellency of these commercial products, registered for use against mosquitoes, against the star tick. The main goal of the present work was to evaluate the effectiveness of six commercial repellents against A. sculptum. Unfed nymphs, aged between two and eight weeks, were tested against products based on DEET (OFF!® and Repelex®), Icaridin (Exposis® and SBP®), and IR3535 (Johnsons and Henlau). Three bioassays were conducted to evaluate tick behavior: (i) filter paper, (ii) fingertip, and (iii) field. All bases tested showed high repellent activity, differing significantly (p < 0.05) from the control. It was observed the formulation with DEET resulted in the best results in the filter paper bioassay. In the fingertip bioassay, the DEET base repelled a greater number of ticks compared to Icaridin. In the field bioassay, there was no significant difference between the Icaridin base and DEET, and both formulations differed from the control (p < 0.05). The six formulations tested showed significant percentages of repellency against ticks; however, for the fingertip and field bioassays, the products OFF!®, Repelex®, and Exposis® were tested as they showed better performance in the filter paper test. OFF!® showed the best percentage of repellency (100%), followed by Repelex® (96.8%), and Exposis® (93.1%), considering the two-hour period of the bioassay-field-test. Proving the effectiveness of repellents on the market against A. sculptum presented in this study is crucial, since this is the main ectoparasite of humans that can transmit Rickettsia rickettsii when infected. The effectiveness of commercial insect repellents against other tick species that parasitize humans can also be explored.

Repellent and acaricidal activity of coconut oil fatty acids and their derivative compounds and catnip oil against Amblyomma sculptum.

This study was carried out aiming to evaluate the repellent and acaricidal activity of major ingredient compounds from coconut oil including their methyl ester derivatives and catnip oil against nymphs and larvae of Amblyomma sculptum. Repellent candidates, coconut oil free fatty acids (coconut FFA mainly C12, C10 and C8 acid); lauric acid (C12 acid); capric acid (C10 acid); methyl laurate; methyl caprate and 10 % each of C12, C10 and C8 acid (1:1:1) in lavender oil formulation (CFA in lavender formula) and catnip oil (Nepeta cataria), were screened using a Petri dish bioassay to assess repellency. Catnip oil, methyl caprate, methyl laurate, and CFA in lavender formulation repelled ticks strongly (P < 0.05) at almost all times evaluated, with an average of 77.8-100% repellency. Some candidate repellents with consistent strong repellence observed were selected for further evaluation, with coconut CFA in lavender formula showing a repellency lasted up to 7 days, while those of catnip oil and methyl caprate were active for 4 and 3 days, respectively. For the acaricide test, five concentrations (2.5; 5; 10; 15 and 20 mg/mL) were evaluated using the larval packet test. Only CFA in lavender formula and two methyl esters showed acaricidal activity, with methyl laurate presenting the strongest toxicity at 15 mg/mL concentration, which was effective against more than 93 % of the tested larvae. Catnip oil caused no mortality of A. scultptum larvae in all concentrations tested.

Seasonal dynamics of Amblyomma sculptum in two areas of the Cerrado biome midwestern Brazil, where human cases of rickettsiosis have been reported.

Amblyomma sculptum is a common human-biting tick in Brazil, where it plays an important role as a vector of Rickettsia rickettsii, the agent of the Brazilian spotted fever. Herein, we studied the seasonal dynamics of A. sculptum in an urban area of the Cerrado biome in midwestern Brazil, where human rickettsiosis is endemic. Ticks were collected in two sites located within the campus of Federal University of Goiás. The collections were done by dragging, flagging and visual search. In total, 117,685 ticks were collected, including 100,627 Amblyomma spp. larvae, 10,055 nymphs and 6977 adults of A. sculptum, and one nymph and 25 adults of Amblyomma dubitatum. The highest peak of larvae occurred in June 2018 and in July 2019, whereas nymphs peaked in July 2018 and September 2019. Adults reached their highest numbers in March 2018 and November 2019. These data suggest that A. sculptum develops one generation per year in this urban area of the Cerrado biome in midwestern Brazil. Interestingly, the peak of nymphs occurred during the same period of all confirmed cases of rickettsiosis in Goiás, suggesting a possible relationship between the seasonal dynamics of this tick stage and rickettsiosis transmission in this state.

Identification of a non-host semiochemical from miniature pinscher, Canis lupus familiaris, that repels Rhipicephalus sanguineus sensu lato (Acari: Ixodidae).

It is already known that the beagle breed of domestic dogs produces semiochemicals capable of repelling the brown dog tick, Rhipicephalus sanguineus sensu lato (s.l.). With a view to discovering new non-host semiochemicals as tick repellents, we compared the semiochemicals produced by a putative tick-resistant breed of dog, miniature pinscher, with known tick-resistant (beagle) and tick-susceptible (English cocker spaniel) breeds. Two non-host compounds produced by beagles, i.e. 2-hexanone and benzaldehyde, were shown to be present in samples collected from all three breeds. Furthermore, two compounds, 6-methyl-5-hepten-2-one and 1,2,4-trimethylbenzene, were found in higher amounts in samples collected from miniature pinscher dogs. The mean amounts of benzaldehyde, 2-hexanone and 1,2,4-trimethylbenzene were similar for beagles and miniature pinschers (P > 0.05) and higher than the means observed for cocker spaniels (P < 0.05), whereas the mean amount of 6-methyl-5-hepten-2-one produced by miniature pinschers was significantly higher (P < 0.05) than for the other breeds of dogs. In Petri-dish assays with adult R. sanguineus s.l., 6-methyl-5-hepten-2-one was repellent for all observation periods evaluated for the two highest concentrations (0.100 and 0.200 mg.cm-2, P < 0.01). The obtained results support our hypothesis that miniature pinschers are a tick-resistant dog breed and agree with previous observations of miniature pinschers being the breed least parasitized by ticks. Furthermore, the non-host semiochemical 6-methyl-5-hepten-2-one has potential to be developed for use as a repellent for the protection of susceptible dogs from R. sanguineus s.l. ticks.

Combination of thymol and eugenol for the control of Rhipicephalus sanguineus sensu lato: Evaluation of synergism on immature stages and formulation development.

The aim of this study was to investigate whether the combination of thymol with eugenol has a synergistic effect on the immature life stages of Rhipicephalus sanguineus sensu lato (s.l.), to evaluate the cost-benefit ratio of using these compounds in combination, and to develop a formulation combining thymol with eugenol with activity on immature stages of R. sanguineus s.l. To evaluate synergism, thymol and eugenol, combined (ratio 1:1) or not, were tested at concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 mg/mL on unfed larvae and nymphs using a larval packet test, and 0.625, 1.25, 2.5, 5.0 and 10 mg/mL on engorged larvae and nymphs using an immersion test. A cost estimate was calculated to produce 1 L of a solution containing a concentration of thymol and eugenol, combined or not, that could cause a tick mortality rate greater than 95 %. Finally, a formulation was developed, consisting of a micellar dispersion containing polymers (MDP), with thymol + eugenol (1:1), at concentrations of 2.5, 5.0, 10.0, 15.0 and 20.0 mg/mL, and the activity was evaluated on unfed and engorged larvae and nymphs. For unfed larvae and nymphs, concentrations of 2.5 and 5.0 mg/mL and 2.5, 5.0 and 10.0 mg/mL, respectively, presented synergistic effects. In tests with engorged larvae and nymphs, respective concentrations of 0.625, 1.25 and 2.5 mg/mL and 2.5 and 5.0 mg/mL had synergistic effects. The estimated costs for producing a solution of 1 L with efficacy greater than 95 % was $5.97 using only thymol (15 mg/mL), $ 5.93 using only eugenol (15 mg/mL), and $ 3.97 using thymol + eugenol (1:1 - 5,0 mg/mL). In tests with MDP, the combination of thymol + eugenol resulted in a mortality rate higher than 95 % at concentration of 10 mg/mL for unfed and engorged larvae and nymphs. Thus, the combination of thymol + eugenol, depending on the concentration, has synergistic effects and this combination lowers the cost for the active ingredients thymol and eugenol. The combination of thymol + eugenol in MDP had acaricidal activity against immature life stages of R. sanguineus s.l.

Thymol: Effects on reproductive biology and Gene's organ morphology in Rhipicephalus sanguineus sensu lato engorged females (Acari: Ixodidae).

Thymol is a monoterpene with proven acaricidal activity on different tick species and life stages. The objective of this work was to evaluate the effect of thymol on the reproductive biology of engorged females of Rhipicephalus sanguineus sensu lato and the morphological changes caused in the cells of the tubular and accessory glands of Gené's organ. The females were exposed to thymol by immersion. Seven groups were established: group I (distilled water), group II (30% ethanol), group III (thymol 1.25 mg/mL), group IV (thymol 2.5 mg/mL), group V (thymol 5.0 mg/mL), group VI (thymol 10.0 mg/mL), and group VII (thymol 20.0 mg/mL), with 20 replicates for each treatment. The ticks were kept in a BOD incubator at 27 ±â€¯1 °C and RH > 80 ±â€¯10%. Ten females from each group were evaluated daily until death, and the remaining ten were kept in the incubator under the same conditions for five days and then dissected for the removal of Gené's organ to note possible damage to cell morphology by scanning electron microscopy (SEM) and hematoxylin-eosin (HE) techniques. Thymol demonstrated 63% and 98% efficacy in groups VI (10.0 mg/mL) and VII (20.0 mg/mL), respectively. In these groups, the cells of the tubular and accessory glands of Gené's organ showed signs of damage: irregular eosin staining, rupture and deformation of the cellular limit, presence of fragmented nuclei, changes in cytoplasmic homogeneity and areas with deformation (folds) in the basal region.

Two for the price of one: Co-infection with Rickettsia bellii and spotted fever group Rickettsia in Amblyomma (Acari: Ixodidae) ticks recovered from wild birds in Brazil.

The bacterium Rickettsia bellii has been detected in 25 species of ticks in the American continents, but its pathogenic potential is considered as undetermined. A possible role for this species in the phenomenon of transovarial exclusion of pathogenic members of the spotted fever group (SFG) of Rickettsia has been suggested and co-infections with pathogenic species have been reported infrequently in both North and South America. Traditional methods for the molecular detection of rickettsial agents in ticks focus largely on the identification of sequences found in SFG Rickettsia, an approach that may overlook the presence of co-infections with R. bellii. Two novel, species-specific polymerase chain reaction (PCR) assays, targeting the genes encoding the surface cell antigen (Sca), autotransporter proteins sca9 and sca14, were developed and validated for the detection of R. bellii using 150 Amblyomma ticks collected from wild birds in Brazil. Co-infection of R. bellii infected ticks was evaluated using a novel PCR assay targeting the ompA sequence characteristic of SFG Rickettsia. Preliminary species-level identification was achieved by restriction fragment length polymorphism (RFLP) analysis and subsequently confirmed by sequencing of amplicons. Nine out of seventy-three Amblyomma longirostre and one of two Amblyomma calcaratum ticks were shown to be co-infected with R. bellii and Rickettsia amblyommatis, while two out of sixty-seven Amblyomma sp. haplotype Nazaré ticks were recorded as co-infected with R. bellii and the Rickettsia parkeri-like bacterium, strain ApPR. Interestingly, our data represent the first records of R. bellii in association with A. calcaratum and Amblyomma sp. haplotype Nazaré. The novel PCR-RFLP systems reported herein, provide an alternative, rapid and cost-efficient (relative to strategies based on sequencing or real-time PCR), approach to evaluate rickettsial co-infection of ticks, a potentially significant phenomenon that has most likely been underestimated to date.

In vitro assessment of the acaricidal activity of carvacrol, thymol, eugenol and their acetylated derivatives on Rhipicephalus microplus (Acari: Ixodidae).

This study reports the comparative evaluation of the activity of carvacrol, thymol, eugenol and their respective acetylated derivatives (carvacrol acetate, thymol acetate and eugenol acetate) on Rhipicephalus microplus, to verify the possible influence of the acetate group. The acetylated derivatives were prepared from reactions of the phenolic compounds with acetic anhydride/pyridine. The formation of the products was confirmed by analysis of hydrogen and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectra. The larval packet test was used to evaluate the acaricidal activity, with concentrations of 0.312, 0.625, 1.25, 2.5, 5.0, 7.5, 10.0 and 15.0 mg/mL. Thymol and carvacrol resulted in 100% mortality starting at the concentration of 2.5 mg/mL, while the same was observed for carvacrol acetate starting at the concentration of 5.0 mg/mL. For the other treatments, 100% mortality was only achieved in the groups treated with the highest concentration (15.0 mg/mL). The LC50 and LC90 values (mg/mL) of carvacrol acetate (2.49, 4.21), thymol acetate (2.97, 8.52) and eugenol acetate (4.25, 13.10) were higher than those for the corresponding precursor molecules carvacrol (0.83, 2.02), thymol (1.26, 2.21) and eugenol (2.77, 5.35). The acetylation process did not enhance the activity of these substances on unengorged larvae of R. microplus, since the precursor substances (carvacrol, thymol and eugenol) had greater efficacy.

Evaluation of the acaricidal activity of thymol incorporated in two formulations for topical use against immature stages of Rhipicephalus sanguineus sensu lato (Latreille, 1806) (Acari: Ixodidae).

The objective of this study was to assess, for the first time, the in vitro acaricidal activity of two topical formulations containing thymol, on immature stages of Rhipicephalus sanguineus sensu lato. For this purpose, two base formulations were prepared: an oil-in-water (O/W) emulsion and a hydroalcoholic solution, containing different thymol concentrations (0.5 to 20 mg/mL). We used the larval packet test for non-engorged larvae and nymphs, and the immersion test for engorged larvae and nymphs. For emulsion, a mortality rate of 94.2% was achieved at 0.75 mg/mL in non-engorged larvae. For engorged larvae, there was 95.0% mortality at 5.0 mg/mL. Non-engorged nymphs showed 83.3% mortality at 2.5 mg/mL, and for engorged nymphs, 86.0% mortality was verified at 5.0 mg/mL. For the hydroalcoholic solution, the mortality found for non-engorged larvae was 88.1% at 2.5 mg/mL. For engorged larvae, the highest mortality was 25.0% at 20 mg/mL; non-engorged nymphs had 91.0% mortality at 1.0 mg/mL and for engorged nymphs; the maximum value verified was 18.3% mortality at 20 mg/mL. Preliminary stability tests were carried, and the hydroalcoholic solution remained stable under all the conditions analyzed. The O/W emulsion showed signs of early instability at the concentration of 5.0 mg/mL. The results obtained indicate that the acaricidal activity of thymol, when included in the proposed formulations, was enhanced against non-engorged larvae with topical treatment in comparison with data in the literature. Although there were variations in toxicity between the different stages, these formulations are promising for future therapeutic use.

Molecular detection of Rickettsia rhipicephali and other spotted fever group Rickettsia species in Amblyomma ticks infesting wild birds in the state of Minas Gerais, Brazil.

The current study evaluated parasitism of wild birds by ticks in a fragment of Atlantic Forest in the state of Minas Gerais, Brazil, and examined the ticks for rickettsial agents. Birds were captured during 2014 and 2015 and ticks were identified by sequencing fragments of the 16S and 12S ribosomal DNA. Among 260 birds representing 19 families and 52 species, a total of 69 (26.5%) were found to be infested by larvae (LL) and/or nymphs (NN) of Amblyomma longirostre (Koch, 1844) (45 LL, 4 NN), Amblyomma calcaratum Neumann, 1899 (9 LL, 15 NN), Amblyomma nodosum Neumann, 1899 (2 NN), Amblyomma parkeri Fonseca and Aragão, 1952 (21 LL), Amblyomma sp. haplotype Nazaré (77 LL), and Haemaphysalis leporispalustris (Packard, 1869) (17 LL, 1 NN). The use of PCR and sequencing of the rickettsial genes gltA, htrA, ompA and ompB, revealed the presence of "Candidatus Rickettsia amblyommii" in A. longirostre (13/49; 26%) and Rickettsia parkeri (strain ApPR) in both A. parkeri (1/21; 5%) and haplotype Nazaré (42/77; 55%) ticks. In addition, we detected Rickettsia rhipicephali in 31 (40%) of the 77 haplotype Nazaré ticks. This is the first record of this rickettsial agent in a species of the genus Amblyomma. The pathogenic potential of this bacterium is undetermined, but the unprecedented association with Amblyomma ticks may represent a cause for concern for public and/or animal health.

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.

New host records of Haemaphysalis leporispalustris (Acari: Ixodidae) on birds in Brazil.

Birds are an important component of the life histories and bioecology of a number of tick species and of some tick associated pathogens. An examination of the data concerning bird/tick associations in the Neotropics, showed that the tick Haemaphysalis leporispalustrisis (Packard, 1869) was rarely recorded infesting birds. The current study reports parasitism by H. leporispalustris in wild birds collected from Atlantic rain forest environments in the states of Rio de Janeiro (4LL) and Minas Gerais (17LL, 1NN), Brazil. All ticks were identified morphologically to the genus level; total DNA was extracted from each Haemaphysalis tick and examined by PCR and nucleotide sequencing of fragments of the eukaryotic genes encoding 16S rRNA and 12S rRNA. The bird species Arremon semitorquatus, Corythopis delalandi, Fluvicola nengeta, Troglodytes musculus, and Volatinia jacarina were recorded as hosts for H. leporispalustris for the first time in South America, and Turdus rufiventris represented a new record for Brazil.

Myrmecofauna of urban gardens in southeast region of Brazil / Mirmecofauna de jardins urbanos na região sudeste do Brasil

In an urban environment, ants can find shelter for the construction of their nests and resources available for survival. Myrmecofauna surveys have been conducted in forest fragments and residential areas, but there are few studies on the occurrence of these insects in urban gardens. For this reason, the aim of this study is to know the composition of ant communities in urban gardens in the city of Juiz de Fora, southeast region of Brazil. Samples were taken from six gardens between November 2005 and June 2006. We used a consortium of three methodologies: active searching, baits and pitfall traps. We collected specimens of 26 genera and seven subfamilies. The subfamily Myrmicinae was the most diverse, and the genus Wasmannia, was considered the most constant, followed by Camponotus and Linepithema. The efficiency index of the methodologies demonstrated that active searching was the most efficient (96.15%) methodology. This method is efficient and sufficient for sampling ants in urban gardens, since 25 of the 26 genera sampled, presenting seven exclusive genera. The green area was not positively correlated with the diversity of ant genera in the urban gardens, demonstrating that other variables may be involved.

Em ambiente urbano, as formigas podem encontrar abrigo para construção de seus ninhos e recursos disponíveis para sobrevivência. Levantamentos da mirmecofauna já foram realizados em fragmentos de mata e áreas domiciliares, porém existem poucos estudos quanto à ocorrência destes insetos em jardins urbanos. Por esta razão, objetivou-se realizar o levantamento da mirmecofauna presente em jardins urbanos do município de Juiz de Fora, sudeste do Brasil. Foram realizadas coletas em seis jardins entre novembro de 2005 e junho de 2006. Foi utilizado o consórcio de três metodologias: busca ativa, isca atrativa e pitfall. Foram coletados exemplares de 26 gêneros e sete subfamílias. A subfamília Myrmicinae foi a mais diversa e o gênero Wasmannia considerado o mais constante, seguido por Camponotus e Linepithema. O índice de eficiência das metodologias demonstrou que a busca ativa foi a metodologia mais eficiente de coleta (96,15%). Este método é eficiente e suficiente para a coleta de formigas em jardins urbanos, uma vez que 25 dos 26 gêneros foram amostrados, com presença de sete gêneros exclusivos. A área verde não esteve correlacionada positivamente com a diversidade de gêneros de formigas nas praças, demonstrando que outras variáveis devem estar envolvidas.

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.

Chemical composition and acaricidal activity of the essential oil of Baccharis dracunculifolia De Candole (1836) and its constituents nerolidol and limonene on larvae and engorged females of Rhipicephalus microplus (Acari: Ixodidae).

Baccharis dracunculifolia DC (common name "alecrim-do-campo" in Brazil) is a plant with widespread distribution in South America that is the botanical origin of green propolis. The aim of this study was to evaluate the chemical composition and acaricidal activity of the essential oil of B. dracunculifolia and its constituents nerolidol and limonene on unengorged larvae and engorged females of Rhipicephalus microplus (Acari: Ixodidae). The essential oil yield was 0.8% of dry mass and the major constituents were nerolidol (22.3%), germacrene D (7.2%), limonene (6.9%), ß-pinene (6.7) and bicyclogermacrene (6.5%). The acaricidal activity of the essential oil and the pure compounds nerolidol and (R)-(+)-limonene were assessed in the laboratory through the modified larval packet test (LPT) and the female immersion test (FIT). In the LPT, the essential oil and nerolidol were both active, causing more than 90% mortality at concentrations from 15.0 and 10.0 mg mL(-1), respectively, whereas (R)-(+)-limonene was not active. In the FIT, the oil and nerolidol caused reduction in the quantity and quality of eggs produced, with control percentages of 96.3% and 90.3% at concentrations of 60.0 and 50.0 mg mL(-1), respectively. It can be concluded that the essential oil obtained from the aerial parts of B. dracunculifolia and its major component nerolidol have high activity on R. microplus larvae and engorged females.

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.

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.

Assessment of the acaricidal activity of carvacrol, (E)-cinnamaldehyde, trans-anethole, and linalool on larvae of Rhipicephalus microplus and Dermacentor nitens (Acari: Ixodidae).

The acaricidal activity of carvacrol, (E)-cinnamaldehyde, trans-anethole, and linalool was studied on Rhipicephalus microplus and Dermacentor nitens larvae. All the substances were tested at concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 µl/ml, with 10 repetitions per treatment. The modified larval packet technique was employed in the tests and the mortality was evaluated after 24 h. In the groups treated with carvacrol, the lowest concentration (2.5 µl/ml) was sufficient to cause 100% death of the R. microplus and D. nitens larvae. The same concentration of (E)-cinnamaldehyde resulted in death of approximately 99% of the larvae of both tick species and reached 100% at the other concentrations. For trans-anethole, mortality rates above 90% of the R. microplus and D. nitens larvae were only observed starting at the concentration of 15.0 µl/ml and reached 100% at the highest concentration (20.0 µl/ml). Finally, the mortality rates of the groups treated with linalool were low, only reaching 8.4 and 14.5% at the highest concentration (20.0 µl/ml) for larvae of D. nitens and R. microplus, respectively. These results show that carvacrol, (E)-cinnamaldehyde, and trans-anethole have acaricidal activity, particularly carvacrol and (E)-cinnamaldehyde, both of which resulted in high mortality rates for the larvae of these two tick species even at the lowest concentration.