Role of Rhipicephalus microplus cheliceral receptors in gustation and host differentiation.

Rhipicephalus microplus is considered the most economically important ectoparasite of cattle worldwide. It is known that zebuine breeds of cattle are less susceptible to tick infestation than taurine breeds. Contact chemoreceptors in the cheliceral pit sensilla of ticks respond selectively to phagostimulant compounds, however their role in blood feeding relative to host susceptibility to infestation remains to be fully understood. We addressed this topic by conducting taste electrophysiology experiments with cheliceral pit sensilla preparations of R. microplus females. Solutions of five known ixodid tick phagostimulants were tested at different concentrations: sodium (NaCl), and potassium chloride (KCl) (10(-3)-10(-1)M); glucose (10(-4)-10(-1)M); adenosine triphosphate (ATP) (10(-6)-10(-2)M); and reduced l-glutathione (GSH) (10(-6)-10(-2)M). Serum samples from six susceptible animals of the Girolando breed (5/8 Bos indicus×3/8 B. taurus) and six resistant Nelore bovines (pure B. indicus) were also tested. A dose-dependent response of gustatory neurons associated with the chelicerae sensillum to NaCl, glucose, GSH, and ATP were observed. Responses by the cheliceral inner digit pit sensilla of R. microplus to KCl and glucose were also observed and they are reported here for the first time. In addition to an electrophysiological response to known phagostimulants, chemoreceptors in the chelicera of R. microplus responded differently to serum from cattle susceptible and resistant to infestation. The cheliceral pit neurons were more responsive to serum of R. microplus resistant bovines with a higher mean spike frequency (53.5±2spikess(-1)) than to serum samples from susceptible cattle (40.3±2spikess(-1)). The implications of chemosensation during tick blood feeding are discussed.

Study on cheliceral sensilla of the brown dog tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) involved in taste perception of phagostimulants.

Phagostimulants are chemical compounds that stimulate feeding. Some tick species are known to have pores in their inner cheliceral digits, called pit sensillum (ps), which are involved in taste perception. This study investigated the existence of ps in chelicerae of Rhipicephalus sanguineus ticks by scanning electron microscopy (SEM) and their involvement in the perception of phagostimulatory substances, such as the salts potassium chloride and sodium chloride (KCl and NaCl), sugars (glucose, sucrose and fructose), purines (guanine and hypoxanthine), the nucleotide adenosine triphosphate (ATP) and the tripeptide reduced glutathione (GSH), as well as their combinations at different concentrations. By means of scanning electron microscopy, a ps was observed at the expected location. Using a single-sensillum recording technique, strong activity by R. sanguineus cheliceral sensilla in response to glucose, ATP, GSH and high concentrations of salts was recorded. The responses to ATP and to KCl at 1M were multicellular, while the responses to the other stimulant compounds were monocellular. Glucose and GSH stimulated different neurons. The taste response of R. sanguineus chelicerae seemed to be selective, given that substances that were not expected to participate in this tick's biology were not perceived.

Electrophysiological responses of the olfactory receptors of the tick Amblyomma cajennense (Acari: Ixodidae) to host-related and tick pheromone-related synthetic compounds.

In the present study, host-related and tick pheromone-related chemical compounds were tested by means of the tip-recording technique in order to obtain electrophysiological responses in olfactory sensilla of non-fed Amblyomma cajennense ticks. The following chemicals were tested on the multiporose sensilla DI.1, located anterior to Haller's organ, and the sensillum DII.1, in the anterior pit of this organ: isobutyric acid, butyric acid, valeric acid, trans-2-heptenal, heptanal, benzaldehyde, salicylaldehyde, nonanal, m-, o- and p-tolualdehyde, 2-furaldehyde, 3-pentanone, γ-valerolactone and 1-octen-3-ol (which are all vertebrate-associated volatiles); and 2,6-dichlorophenol (2,6-DCP), 2-nitrophenol, methyl salicylate and nonanoic acid (tick pheromone components). These were used at 10(-3)M and 10(-2)M on at least 10 ticks per substance, and the chemicals that were found to be active at these concentrations were then tested as a series from 10(-6)M to 10(-2)M, in decadic steps, on at least 15 ticks per substance. 2,6-DCP was active on both sensilla, with detection thresholds of 10(-6)M on the DI.1 sensillum and 10(-4)M on the DII.1 sensillum. The olfactory neurons of this sensillum also responded to nonanal at the highest concentration used (10(-2)M), while those of DII.1 responded not only to 2.6 DCP but also to 2-nitrophenol (to the same extent as to 2,6-DCP) and to 1-octen-3-ol. These results confirm the importance of 2,6-DCP in the chemical ecology of A. cajennense and indicate other compounds that may interfere with the behavior of this tick and which should be investigated.

Potential synergistic effect of Melia azedarach fruit extract and Beauveria bassiana in the control of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) in cattle infestations.

The use of a concentrate emulsion of Melia azedarach green fruits and a suspension of the fungus Beauveria bassiana was evaluated in the control of Rhipicephalus microplus on artificially infested cattle. The evaluation was conducted following the protocol established by the Brazilian Agriculture Ministry. Five groups of 4 or 5 animals were allocated to one of the following treatments: emulsion concentrate of M. azedarach at 0.25% (T AZED 0.25%), emulsion concentrate of M. azedarach at 0.5% (T AZED 0.5%), B. bassiana at 2.4 × 10(8) conidia (T BASS), association of the concentrate of M. azedarach at 0.25% with B. bassiana at 2.4 × 10(8) conidia (T AZED 0.25%+BASS), and control (untreated). The association of the two compounds provided better results than any one isolated treatment, indicating compatibility or perhaps a synergy between M. azedarach and B. bassiana. This treatment resulted in fewer engorged females (129 ± 70) than in the control group (233 ± 82), showing high performance against all developmental stages of the tick. Results revealed an apparent synergistic effect of M. azedarach and B. bassiana in the control of R. microplus that should be further investigated.

Repellent activity of DEET against Amblyomma cajennense (Acari: Ixodidae) nymphs submitted to different laboratory bioassays.

This study was developed to evaluate the repellent activity of N,N-diethyl-3-methylbenzamide (DEET) against Amblyomma cajennense nymphs. Two repellent bioassays were compared and the effective concentration and repellent time were calculated. The fingertip test was accomplished to evaluate in vivo four concentrations of the compound (0.200; 0.100; 0.050 and 0.025 mg.cm⁻²) and the filter-paper bioassay to evaluate in vitro the two highest concentrations.The compound provided repellence higher than 90% in all concentrations and at least 95% repellency in the highest concentration over 5 hours. The effective concentration against 50% of tested nymphs (EC50) was 0.006 mg.cm⁻² and the EC99 was 0.036 mg.cm⁻². Those concentrations were lower than the ones obtained against other tick species, denoting the effectiveness of DEET against A. cajennense. The repellency time against 50% of the ticks (RT50) was 4.8 hours and the RT90 was 2.7 hours. Both bioassays were adequate to evaluate A. cajennense repellency and provided similar results; however the in vivo test is more appropriate to estimate the effective concentration and repellency time.

Repellent activity of DEET against Amblyomma cajennense (Acari: Ixodidae) nymphs submitted to different laboratory bioassays / Atividade repelente do DEET contra ninfas de Amblyomma cajennense (Acari: Ixodidae) em bioensaio laboratorial

This study was developed to evaluate the repellent activity of N,N-diethyl-3-methylbenzamide (DEET) against Amblyomma cajennense nymphs. Two repellent bioassays were compared and the effective concentration and repellent time were calculated. The fingertip test was accomplished to evaluate in vivo four concentrations of the compound (0.200; 0.100; 0.050 and 0.025 mg.cm-2) and the filter-paper bioassay to evaluate in vitro the two highest concentrations. The compound provided repellence higher than 90 percent in all concentrations and at least 95 percent repellency in the highest concentration over 5 hours. The effective concentration against 50 percent of tested nymphs (EC50) was 0.006 mg.cm-2 and the EC99 was 0.036 mg.cm-2. Those concentrations were lower than the ones obtained against other tick species, denoting the effectiveness of DEET against A. cajennense. The repellency time against 50 percent of the ticks (RT50) was 4.8 hours and the RT90 was 2.7 hours. Both bioassays were adequate to evaluate A. cajennense repellency and provided similar results; however the in vivo test is more appropriate to estimate the effective concentration and repellency time.

Este estudo foi conduzido com o objetivo de avaliar a atividade repelente do N,N-diethyl-3-methylbenzamide (DEET) sobre ninfas de Amblyomma cajennense. Dois bioensaios para a avaliação de repelência foram comparados e cálculos da concentração eficaz e do tempo de repelência foram realizados. Foram empregados o bioensaio da ponta do dedo, para avaliação in vivo de quatro concentações do químico (0,200; 0,100; 0,050 e 0,025 mg.cm-2) e o bioensaio do papel filtro, para a avaliação in vitro das duas concentrações mais altas. O composto conferiu mais de 90 por cento de repelência em todas as concentrações utilizadas e 95 por cento de repelência por mais de cinco horas na maior concentração. A concentração do composto efetiva contra 50 por cento das ninfas testadas (CE50) foi de 0,006 mg.cm-2 e a CE99 foi de 0,036 mg.cm-2. Estas concentrações são mais baixas do que as observadas em outras espécies de carrapatos, denotando a efetividade do princípio contra A. cajennense. O tempo de repelência de 50 por cento dos carrapatos (TR50) foi de 4,8 horas e o TR90 de 2,7 horas. Os dois bioensaios avaliados permitiram a observação de percentuais de repelência igualmente altos e se mostraram adequados para tal avaliação, sendo que o teste in vivo é mais indicado para cálculo da concentração eficaz e da duração da repelência.

Differences in the behavior of Rhipicephalus sanguineus tested against resistant and susceptible dogs.

To ascertain whether brown dog tick Rhipicephalus sp. infests resistant (beagle) and susceptible (English cocker spaniel) dogs differently, five animals of each breed were maintained in a kennel whose walls were infested with 7,000 larvae, 320 nymphs, 80 males and 80 females, in 3 infestations, at 10-day intervals. Five times more ticks were found on cocker spaniels (498) than on beagles (96). Substances were collected by rubbing pieces of clean flannel on the dogs for 15 min and these were tested for arrestment and attractiveness of ticks. Three choices were offered: cocker extract vs. control; beagle extract vs. control, and cocker extract vs. beagle extract. When allowed to choose between substances rubbed from dogs and a control, more ticks were arrested by extracts from the cockers than from beagles. In the arrestment tests with only a choice between substances from dogs of each breed, more ticks were arrested by cocker substances. To test for attraction, capsules containing adsorbent were used and the tests were carried out in a Y-olfactometer. Fifteen males and 15 females were tested, for each treatment. In the olfactometer, the ticks were not attracted to the odor of either breed, however the odor of the Beagle was apparently repellent. These results indicate that R. sanguineus can use substances from the dogs to differentiate susceptible English Cocker Spaniels from resistant Beagles.

Repellent activity of plant-derived compounds against Amblyomma cajennense (Acari: Ixodidae) nymphs.

Repellence responses of Amblyomma cajennense nymphs to callicarpenal, intermedeol, Hyptis suaveolens essential oil, extract of Melia azedarach, Cymbopogon nardus, Spiranthera odoratissima, Chenopodium ambrosioides, Ageratum conyzoides, Mentha pulegium, Ruta graveolens, and Memora nodosa were studied. Among these the extract of C. nardus stood out because of the long-lasting repellence, maintaining, in the highest concentration, 35h of protection against 90% of the nymphs. The essential oil of H. suaveolens and the extracts of C. ambrosioides and A. conyzoides showed good repellence index (66%) when applied in high concentrations. However, greater protection could be obtained at higher concentrations but with a shorter repellence time. Callicarpenal, intermedeol, extract of M. Pulegium, and M. nodosa leaves showed moderate repellence in high concentrations. Extracts from M. azedarach, R. graveolens, S. odoratissima, and M. nodosa roots showed little or no repellent effect. These results show that some plant extracts may represent a promising alternative in the control of infestations by A. cajennense.

More about the role of 2,6-dichlorophenol in tick courtship: identification and olfactometer bioassay in Amblyomma cajennense and Rhipicephalus sanguineus.

This study aimed to identify 2,6-dichlorophenol (2,6-DCP) in Amblyomma cajennense and to evaluate its role in A. cajennense and Rhipicephalus sanguineus courtship. Hexanic extract from attractive females was purified by solid phase extraction and the phenol was identified by the single ion monitoring method using GC/MS. In an olfactometer, the responses of A. cajennense and R. sanguineus males to females, control rubber septa or rubber septa impregnated with 2,6-DCP at 50, 500, and 5000 ng, respectively, were studied. 2,6-DCP was identified in A. cajennense extract and the males oriented themselves toward the concentration of 500 ng. These septa and the females were recognized as copula partners. The septa treated with 2,6-DCP did not attract and were not even recognized by the R. sanguineus males, whereas the females were recognized. Due to the presence of 2,6-DCP in A. cajennense and the results of biological bioassays, it was concluded that this compound acts as an attractant and mounting sex pheromone in this tick, but it does not play any role in R. sanguineus courtship.

More about the role of 2,6-dichlorophenol in tick courtship: identification and olfactometer bioassay in Amblyomma cajennense and Rhipicephalus sanguineus

This study aimed to identify 2,6-dichlorophenol (2,6-DCP) in Amblyomma cajennense and to evaluate its role in A. cajennense and Rhipicephalus sanguineus courtship. Hexanic extract from attractive females was purified by solid phase extraction and the phenol was identified by the single ion monitoring method using GC/MS. In an olfactometer, the responses of A. cajennense and R. sanguineus males to females, control rubber septa or rubber septa impregnated with 2,6-DCP at 50, 500, and 5000 ng, respectively, were studied. 2,6-DCP was identified in A. cajennense extract and the males oriented themselves toward the concentration of 500 ng. These septa and the females were recognized as copula partners. The septa treated with 2,6-DCP did not attract and were not even recognized by the R. sanguineus males, whereas the females were recognized. Due to the presence of 2,6-DCP in A. cajennense and the results of biological bioassays, it was concluded that this compound acts as an attractant and mounting sex pheromone in this tick, but it does not play any role in R. sanguineus courtship.

Efficacy of 2,6-dichlorophenol lure to control Dermacentor nitens.

This study was carried out with the objective of evaluating the efficacy of a 2,6-dichlorophenol (2,6-DCP) lure to control Dermacentor nitens (Acari: Ixodidae). Slow-release formulations of the pheromone formulated with and without cypermethrin were prepared. Olfactometer bioassays were used to define the best dose of the pheromone and to evaluate the effect of cypermethrin with 2,6-DCP attractiveness. Sexually active males were released 15 cm from 2 cmx1 cm pieces of polypropylene treated with different odors: 2,6-DCP in a liposphere system (1.5, 30 and 300 microg--without cypermethrin and 30 microg--with cypermethrin) and 2,6-DCP in hexane (30 microg). The tests were conducted 7 and 15 days after the preparation of the odor sources. The percentages of males that oriented, or showed directional movement toward the stimulus, and their tracks were recorded. Lures (10 cmx2 cm pieces of polypropylene) treated with the best dose of the liposphere formulation (30 microg) were prepared. The lures were evaluated in horses that had been artificially infested with D. nitens. The horses' ears were infested with 3000 D. nitens larvae per ear, once weekly for 4 weeks. The animals were divided into three groups: control, 2,6-DCP and 2,6-DCP+cypermethrin. On day 0, the lures of their respective treatments were attached to the horses' napes. From days 6 to 20 after attachment, female ticks of 4 mm or over in length were counted on the ears, every 2 or 3 days. Olfactometer analysis showed higher orientation to 30 microg dose and more prolonged release of the pheromone in the liposphere formulation than in hexane; cypermethrin did not interfere with the attractive effect of the pheromone. The lures were efficient in the first 10 days after attachment, when the mean number of females was higher in the control group (24.9) than in 2,6-DCP and cypermethrin (5.4), and 2,6-DCP (9.2) groups. After that period the number of females was similar in the control and treated groups. These results indicate that 2,6-DCP lures used in a liposphere formulation can control D. nitens for at least 10 days by preventing its copulation. However, further evaluation of longer-term pheromone release under natural conditions is needed in order to validate this kind of control. In addition, the use of extra lures on the horse's tail may help to control populations on the hindquarters.

Resistência acaricida em larvas de Rhipicephalus sanguineus (Acari: Ixodidae) de Goiânia-GO, Brasil / Acaricide resistence in Rhipicephalus sanguineus (Acari: ixodidae) larvae from Goiânia-GO, Brazil

Fêmeas ingurgitadas de Rhipicephalus sanguineus foram colhidas em cães provenientes de diferentes regiões de Goiânia e incubadas para a produção de larvas. Usando-se um teste de imersão, larvas de 7 a 21 dias de idade foram testadas ante os seguintes acaricidas e concentrações (em ppm): amitraz (250, 125, 62,5 e 31,2), coumafós (500, 250, 125 e 62,5), cipermetrina (150, 75, 37,5 e 18,7), deltametrina (25, 12,5, 6,2 e 3,1) e fipronil (312, 156, 78, 39 e 19,5). Cálculos das DL50 e DL99 foram feitos por meio do programa de análise do probito. Cepas com DL99 acima daquelas recomendadas pelo fabricante ou pela literatura foram consideradas como resistentes. Nenhuma das 15 amostras testadas ante o amitraz teve a DL99 superior à concentração recomendada. Ante a deltametrina, a cipermetrina e o coumafós, nove (75por cento), sete (78por cento) e sete (78por cento) amostras, respectivamente, tiveram DL99 superior à recomendada. As DL50 variaram de 4 a 16 ppm, 0,3 a 3,2 ppm, 1 a 20 ppm e 17 a 75 ppm em relação a amitraz, deltametrina, cipermetrina e coumafós, respectivamente. Houve mortalidade de 100por cento das larvas testadas em todas as concentrações do fipronil. Estes resultados demonstram que a maioria das cepas de R. sanguineus de Goiânia apresentaram resistência à cipermetrina, à deltametrina e ao coumafós. Não se verificou resistência ao amitraz na população de R. sanguineus estudada.

Ação do extrato hexânico de frutos maduros de Melia azedarach (Meliaceae) sobre Boophilus microplus (Acari: Ixodidae) em bezerros infestados artificialmente / Efficacy of Melia azedarach (Meliaceae) hexanic ripe fruit extract against Boophilus microplus (Acari: Ixodidae) on artificially infested calves

A eficácia de um extrato hexânico dos frutos de Melia azedarach foi avaliada em bezerros infestados artificialmente com Boophilus microplus. Os frutos foram submetidos ao processo de secagem e, depois de moídos, o extrato foi produzido em Soxhlet utilizando-se o hexano como extrator. Oito bezerros infestados artificialmente, cinco vezes a cada cinco dias com 2.500 larvas de B. microplus, foram divididos em dois grupos (tratado e controle) com base na infestação por carrapatos. No dia zero os animais foram banhados com 3 litros de uma solução a 0,25por cento do extrato em água e acetona (1,2 litro). Os animais do grupo controle foram banhados com o mesmo volume de água e acetona. Diariamente, as fêmeas ingurgitadas desprendidas naturalmente eram contadas e 40 eram incubadas para acompanhamento dos parâmetros reprodutivos. O número médio de fêmeas, o índice de conversão em ovos (peso da massa de ovos/ peso do grupo de fêmeas x 100) e o percentual de eclosão, obtidos nos dois grupos, foram comparados mediante o teste T de Student. Após 21 dias de contagens, o número médio de fêmeas ingurgitadas foi significativamente menor (P<0,05) no grupo tratado (188) do que no controle (247). Porém, a conversão e a eclodibilidade das larvas não foram afetadas pelo tratamento. Estes resultados indicam que houve interferência da planta no desenvolvimento do carrapato sobre os animais, mas não na sua reprodução. Comparados com a literatura, os resultados se revelaram promissores. No entanto, novos estudos nos quais se empregue uma formulação adequada em uma concentração mais elevada são necessários para se propor o uso desta planta no controle deste carrapato.