Carla Cristina Braz Louly

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.

Seasonal dynamics of Rhipicephalus sanguineus (Acari: Ixodidae) in dogs from a police unit in Goiânia, Goiás, Brazil

The seasonal dynamics of Rhipicephalus sanguineus ticks was developed in dogs from a Police Unit in Goiânia, Goias, Brazil, from July 2001 to July 2002. The study was carried out on seven naturally infested dogs (two English Cocker Spaniels and five mongrel dogs), with ages between six months and 10 years. Every two weeks, the numbers of feeding larvae, nymphs, and adults were determined. Dogs showing infestation levels above 500 adult ticks received three acaricide treatments. Considering that the treatments had affected the development of some peaking populations of ticks, it was inferred the occurrence of the following peaks: - larvae (four peaks): from August to November, from November to February, from March to May, and from May to July; - nymphs (five peaks): from July to September, from October to December, from December to February, from March to May, and from June to July; - adults (four peaks): from July to October, from October to January, from January to March, and from April to July. The occurrence of these consecutive peaks of activity of each stage of R. sanguineus may indicate that this tick can develop up to four generations per year in Goiânia. On the other hand, if the acaricide treatment did not interfere with the development of R. sanguineus peaks, more than four peaks of each stage have occurred on the dogs. In this case, it is acceptable to infer that more than one population of R. sanguineus was developing within the kennel concomitantly. The mean numbers of each tick stage was similar in the different seasons. The main attachment sites were located on the neck, chest, forelegs, armpits, ears, between toes and on the head. The number of adult ticks feeding on English Cocker Spaniel dogs was 1.4 to 11.5 times higher than that feeding on mongrel dogs.

Differences in the susceptibility of two breeds of dogs, English cocker spaniel and beagle, to Rhipicephalus sanguineus (Acari: Ixodidae)

The objective of this study was to evaluate differences in susceptibility to Rhipicephalus sanguineus (Latreille) of two breeds of dogs: English cocker spaniel and beagle. Three artificial infestations of ticks using a capsule on the back of the dogs were carried out at 15 day intervals. Six dogs of each breed were infested with 100 larvae, 50 nymphs and 20 adults (10 males and 10 females). Every 2 days, the ticks on the dogs were evaluated and the engorged ones counted and collected. The ticks were stored in climatic chambers and inspected every two days for 14 days. The following parameters were recorded: number of engorged ticks, engorgement period, percentage of ecdysis, female weight, conversion of body weight to eggs and larval hatchability. In the ticks recovered from the cocker spaniel, higher percentages of ecdysis of larvae and nymphs, greater number of adults attached and engorged and higher reproductive performance of the females (conversion to eggs and hatchability) were observed. The period of engorgement, number of engorged nymphs and weight of the females did not differ much by breed, but more larvae were attached and engorged in the beagle. In the two breeds, an effect of the successive infestations on the number of engorged larvae and nymphs was observed, which demonstrates the development of a response to the infestation in these stages. However, the percentage of ecdysis and the conversion to eggs was affected only in the more resistant beagles. Based on what was presented, it can be concluded that the animals of the race English cocker spaniel are more susceptible to R. sanguineus than the beagles and that the infestations affect the development of the tick, especially in resistant individuals.

Identification of non-host semiochemicals for the brown dog tick, Rhipicephalus sanguineus sensu lato (Acari: Ixodidae), from tick-resistant beagles, Canis lupus familiaris

Studies have shown that the brown dog tick, Rhipicephalus sanguineus sensu lato, when fed on the beagle breed of dog, Canis lupus familiaris, development negatively affected in comparison with tick development after feeding on the English cocker spaniel breed. Thus leading to the suggestion that beagle dogs are be tick-resistant dogs. Behavioural studies have demonstrated that more ticks are attracted by extracts from cocker spaniels than from beagles and that the odour of beagles is a repellent. To test the hypothesis that resistant hosts produce repellent compounds, we undertook comparative chemical analysis on beagle odour and cocker spaniel extracts using coupled high-resolution gas chromatography-mass spectrometry (GC-MS) and also used Petri-dish and olfactometer behavioural assays to assess the response of ticks to identified non-host compounds. The beagle odour extracts contained almost three times as many chemical compounds as cocker spaniel samples. Several non-host compounds were identified, i.e. 2-hexanone, benzaldehyde, nonane, decane and undecane. In Petri-dish assays, 2-hexanone was repellent at 30 min at concentrations of 0.200 and 0.050 mg cm(-2), whilst at 10 min, the 0.100 mg cm(-2) concentration was repellent. Benzaldehyde repelled ticks at 30 min (0.200 mg cm(-2)) and at 5 min (0.050 mg cm(-2)). Undecane was repellent for R. sanguineus s.l. ticks for the first 5 min at the highest concentration tested. Nonane and decane did not show any significant repellency at any concentration or time evaluated. When 2-hexanone and benzaldehyde were combined, an increase in the repellency rate was observed, with activity comparable or better than N,N-diethyl-3-methylbenzamide (DEET). In olfactometer bioassays, a 1:1 mixture of 2-hexanone:benzaldehyde and DEET were repellent for R. sanguineus s.l. adults at the concentration of 0.200 mg cm(-2). This study identified non-host semiochemicals that mediate avoidance of the beagle dog breed by R. sanguineus s.l. This finding may enable development of new approaches to control this tick.

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.

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 ticks biology were not perceived.

Induction of complete courtship ritual in Amblyomma cajennense using 2,6-dichlorophenol at female-equivalent quantities

In order to clarify the role of 2,6-dichlorophenol (2,6-DCP) in the courtship of Amblyomma cajennense, sexually mature males that had previously fed on rabbits were tested in bioassays. The males were released onto dummies treated with whole female extract or synthetic 2,6-DCP at a concentration of two female equivalents, or with hexane (control), and their responses were observed. In the presence of both the extract and 2,6-DCP, excitation was observed among the males, expressed in the form of touching and probing the dummy, and mounting occurred readily. The percentages of mounting (73%) and tipping over (60%) were equal in the two treatments and higher than in the control group (27 and 20%, respectively). Relatively short durations of mounting were recorded, and these were statistically similar in all treatments. Almost all instances of mounting resulted in tipping-over behavior. A few isolated cases of males that went directly to ventral positioning without mounting were observed. It was confirmed that 2,6-DCP alone is capable of mediation of mounting behavior in A. cajennense.

Comparison of two methods to quantify 2,6-dichlorophenol from tick Amblyomma cajennense by GC/MS-SIM

Amblyomma cajennense (Acari: Ixodidae) e um carrapato de grande importância socioeconomica no subcontinente Sul-Americano. Apesar disso, pouco se conhece acerca de sua ecologia quimica, cuja informacao e crucial para o seu controle. Nesse estudo, 2,6-diclorofenol (2,6-DCP), o feromonio sexual de A. cajennense foi quantificado por cromatografia gasosa acoplada a espectrometria de massas no modo de analise por monitoramento seletivo de ions (CG/EM-MSI) a partir de femeas alimentadas em coelhos durante 6 dias. O extrato do feromonio sexual foi obtido pela exposicao ao ultrassom de femeas virgens em hexano em duas amostras independentes. Nenhum pre-tratamento da amostra foi necessario. Os metodos de adicao padrao (SA) e de curva de calibracao com 5-bromo-4-hidroxi-3-metoxibenzaldeido (5-BrV) como padrao interno (IS) foram utilizados para a quantificacao. O conteudo de 2,6-DCP nao apresentou diferenca significativa entre os extratos e/ou metodos utilizados. Os resultados mostraram que as faixas de concentracao de 2,6-DCP por femea foram de 2,03-2,27 ng mL -1 e de 2,06-2,24 ng mL -1 para os metodos SA e IS, respectivamente. Os metodos mostraram ser especificos, sensiveis e fidedignos na determinacao de 2,6-DCP em carrapatos.

Detection of phytoecdysteroids by gustatory sensilla on chelicerae of the brown dog tick Rhipicephalus sanguineus

Ecdysteroids are polyhydroxylated steroids that act as moulting hormones in arthropods and regulate several important life‐cycle processes. Phytoecdysteroids are ecdysteroid analogues produced by some plants that disrupt the growth and development of insects feeding on them, and can be perceived by the taste receptors of insects. The present study tested the hypothesis that the blood‐feeding tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) is capable of detecting phytoecdysteroids. By recording from the chelicerae, six phytoecdysteroids are tested: α‐ecdysone, 20‐hydroxyecdysone, ponasterone A, makisterone A, inokosterone and pterosterone. In unfed ticks, makisterone A and pterosterone elicit frequencies of neural impulses higher than in a negative control (a KCl solution at 10−3m), with detection thresholds of 10−6m and 10−12m, respectively. The spike amplitudes of the responses to these compounds, and also for 20‐hydroxyecdysone and ponasterone A, are higher than in the control, indicating that a different neurone may be involved: perhaps a deterrent cell, as observed in insects. In fed ticks, only pterosterone at 10−4m remains active. In behavioural attachment assays, no difference is observed between electrophysiologically active compounds and the negative control. These results show the capability of R. sanguineus ticks to detect phytoecdysteroids, although they do not clarify the role of ecdysteroids in tick biology, for which further studies are required.

Chemical composition and biological characterization of extracts of male-produced pheromone from Amblyomma cajennense (Fabricius, 1787) (Acari: Ixodidae) by GC/MS-SIM and behavioural responses

The extract from fed Amblyomma cajennense (Fabricius) males was characterized for chemical composition and behavioural responses. The pheromone was obtained by extraction in hexane, analysed by gas chromatography/mass spectrometry-single ion monitoring and subjected to behavioural bioassays in vitro and in vivo. The presence of 10 constituents already identified in tick pheromones was evaluated and qualitative analysis of the extract indicated the presence of benzaldehyde, benzoic acid, nonanoic acid, salicylic acid, 2,6-dichlorophenol, limonene and methyl salicylate and the absence of butyric acid, isobutyric acid and o-nitrophenol. In an olfactometer bioassay, males, females and nymphs were not attracted by the extract (P > 0.05) as the displacement angles (0°–10°) distribution was similar between tests (13.3–38.6%) and controls (8.5–23.7%). Similarly, there was no statistically significant aggregation response of test ticks to extracts in bioassays in vitro. However, significantly greater attachment (P < 0.05) in tests with extracts was registered within a 24-h period than that in the control in males but not in females. A synthetic simulation, constituted according to the proportions obtained in the chemical analysis, yielded comparable results. The behavioural observations made during the study were consistent with the extracts chemical composition. The potential implication of the findings in the management of A. cajennense in the regions where it is endemic is discussed.