Repellent activity of the non-host semiochemical (E)-2-octenal against Amblyomma sculptum and Amblyomma dubitatum ticks under field conditions.

Amblyomma ticks pose a significant public health threat due to their potential to transmit pathogens associated with rickettsial diseases. (E)-2-octenal, a compound found in donkeys (Equus asinus), exhibits strong repellent properties against Amblyomma sculptum nymphs under laboratory conditions. This study assessed the effectiveness of the (E)-2-octenal in wearable slow-release devices for personal human protection against Amblyomma ticks under natural conditions. Slow-release devices treated with (E)-2-octenal and untreated controls were prepared and tested on two volunteers walking through a tick-infested area in Goiania, Brazil. The experiment was conducted twice daily for three series of 10 days, with each volunteer wearing two devices attached to each leg, one on the ankle and one just above the thigh. Volunteers with control and treated devices exchanged them between rounds. Also, the daily release rate of (E)-2-octenal from the slow-release devices was determined in the laboratory, increasing significantly from 0.77 ± 0.14 µg/day on the first day to 9.93 ± 1.92 µg/day on the 4th day and remaining constant until the 16th day. A total of 5409 ticks were collected from both volunteers. Treated devices resulted in recovering fewer ticks (n = 1,666; 31%) compared to untreated devices (control: n = 3,743; 69%). (E)-2-octenal effectively repelled Amblyomma spp. larvae, A. sculptum adults, and exhibited pronounced repellency against A. dubitatum nymphs and adults. These findings suggest the potential of (E)-2-octenal delivered by wearable slow-release devices as a green-based repellent. Further improvements, however, are necessary to provide better protection for humans against A. sculptum and A. dubitatum in field conditions.

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.

In vitro activity of 3ß-O-tigloylmelianol from Guarea kunthiana A. Juss (Meliaceae) on oogenesis and ecdysis of the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae).

We evaluated the effects of 3ß-O-tigloylmelianol from Guarea kunthiana A. Juss (Meliaceae) on oogenesis, as a larvicide and on ecdysis of the larvae and the nymphs of the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) (Acari: Ixodidae). On the oogenesis' test, 48 engorged females were divided into three groups, evaluated at 24, 48 and 72 h post-treatment. Half of the females were treated with 0.01% 3ß-O-tigloylmelianol diluted in distilled water and 5% dimethyl sulfoxide (DMSO), while the other half (controls) were exposed to distilled water and 5% DMSO. After treatment, the ovaries were weighed in order to measure the gonadosomatic index (GSI) and were also subjected to standard histological technical tests. On the larvicide and ecdysis' tests, 3ß-O-tigloylmelianol was tested at concentrations of 0.01, 0.005, 0.0025 and 0.00125%. Compared with the controls, there was a reduction of GSI of approximately 50% on the treated group, which started at 48 h post treatment. Overall, the protolimonoid 3ß-O-tigloylmelianol has caused a significant reduction in the number of oocytes. It has also caused alteration of the cytoplasmic and germinal vesicle diameters. Morphological changes, such as vacuolization, chorion irregularity which has modified the oocytes' morphology as well as alterations on the yolk's granules were also observed. The compound was not larvicide, however, interfered in the ecdysis of the larvae and the nymphs. This study shows that the protolimonoid 3ß-O-tigloylmelianol from G. kunthiana acts on oogenesis and ecdysis of R. (B.) microplus, but not as larvicide, indicating that it acts on the endocrine system of the tick.

In vitro activities of plant extracts from the Brazilian Cerrado and Pantanal against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

A total of 73 ethanol extracts from different anatomical parts of 44 plant species belonging to 24 families, native to the Mid-Western region of Brazil, were assessed in vitro for their effect on the reproductive cycle of engorged females of the cattle tick Rhipicephalus (Boophilus) microplus, using the adult immersion test. All extracts were evaluated at the concentration of 0.2 % and, among the extracts tested, the one obtained from the fruits of Guarea kunthiana (Meliaceae) proved to be highly efficacious, showing 99.1 % of product effectiveness. Extracts from other three species were shown to be moderately active, namely Nymphaea amazonum trunk (Nymphaeaceae) [51.7 %], Strychnos pseudoquina trunk (Loganiaceae) [48 %] [corrected] and Ocotea lancifolia leaves (Lauraceae) [34.5 %], while the remaining extracts were shown to be weakly active or inactive. This is the first report on the bioactivity of these species on egg production by engorged females of R. microplus.

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.

First neuronal projection from Haller's organ to the synganglion and three-dimensional reconstruction of Amblyomma sculptum olfactory lobe.

Because information regarding neural and sensory functions of ticks is scarce, the aim of the present study was to ascertain the neuronal projection patterns of olfactory sensilla from Haller's organ to the olfactory lobes of the synganglion in Amblyomma sculptum adults. Additionally, the aim was to perform three-dimensional (3D) reconstruction of the glomeruli in the olfactory lobes. Unfed female and male adults of this tick species were immobilized, Haller's organ was sectioned, and the olfactory nerves were labelled with a neuronal tracer (1% dextran tetramethylrhodamine). The labelled olfactory lobes were observed under a confocal laser scanning microscope and the images were analyzed using the Reconstruct software. The neuronal projection originating from the olfactory sensilla of Haller's organ was found to be strictly confined to the olfactory lobes of the synganglion, which were organized into glomeruli. Males and females presented corresponding round-shaped glomeruli in the olfactory lobes. The number of glomeruli ranged from 26 to 30 in the males and 24-29 in the females. Positive correlations regarding the glomeruli volumes (µm3) intrasexually (females: r = 0.85, P < 0.05) and intersexually (r = 0.81, P < 0.05) were observed. This is the first report on 3D reconstruction of the olfactory lobes of a tick species.

Thymol and eugenol microemulsion for Rhiphicephalus sanguineus sensu lato control: Formulation development, field efficacy, and safety on dogs.

The present study aimed to develop a microemulsion formulation containing thymol and eugenol for field control of Rhipicephalus sanguineus sensu lato on dogs, as well to evaluate its safety and the physical characteristics of the formulation. The microemulsion using thymol and eugenol (5.0 + 5.0 mg/mL) had as vehicles water, propylene glycol, polysorbate 80 and canola oil. On the next day the preparation (formulation freshly prepared) and after 24 months, the size of the microemulsion droplets, polydispersion index (PdI), organoleptic properties (color, viscosity), and presence of precipitate in the microemulsion were evaluated. For the field assay, on day -1, 10 English Cocker Spaniel dogs were experimentally infested with 200 larvae, 100 nymphs and 30 adults of R. sanguineus s.l. On day 0, after tick counts, the animals were divided into two groups: treated with the freshly prepared microemulsion (10 mL/kg), and control, which received the vehicle (10 mL/kg). Tick counts on dogs were performed daily for three more days. Engorged females were recovered from the dogs and their biological and reproductive parameters were monitored. The dogs' clinical parameters (temperature, mucosa color, and general physical condition) were evaluated daily. In addition, blood samples were collected before infestation to verify hematological (packed cell volume) and biochemical parameters (total serum protein, albumin, globulins, creatinine, urea, alanine transaminase, aspartate aminotransferase, and alkaline phosphatase). Freshly prepared and 24-month aged microemulsions had droplets with mean sizes of 30.94 nm and 27.93 nm, and PdI values of 0.214 and 0.161, respectively. In addition, no difference in the organoleptic properties and no precipitation formation were observed, indicating physical stability. Treatment with the microemulsion resulted in reduction of larvae (p < 0.05) parasitizing the dogs on day 1 while the number of nymphs and adults was not reduced (P> 0.05). In the evaluation of the reproductive biology of engorged females, the larval hatchability (%) was compromised (p < 0.05), and the microemulsion had control rate of 85.5 %. The microemulsion and its vehicles did not change the clinical, hematological and biochemical parameters of the dogs. We concluded that the microemulsion was efficient against R. sanguineus s.l. by reducing the number of larvae and affecting the reproductive parameters of engorged females, safe for dogs, and stable (physical stability) during a two-year interval.

Rhipicephalus microplus seasonal dynamic in a Cerrado biome, Brazil: An update data considering the global warming.

The present study evaluated over two years the seasonal dynamics of Rhipicephalus microplus in a Cerrado biome of midwestern Brazil (Goiânia, Goiás) and correlated the current climatic conditions (environmental temperature, insolation, rainfall, relative humitidy and saturation deficit of the atmosphere) of this site with 30 years ago. In addition, the data collected in the present research were compared with climatic conditions data and R. microplus population dynamics conducted by our group in other regions (Formiga, Minas Gerais and Jaboticabal, São Paulo) within the same biome. For the parasitic phase dynamics, R. microplus females (4.5-8.0 mm) counts were performed on tick natural infested cows kept in Panicum maximum paddocks. To verify the larvae dynamics on pasture the flannel dragg technique on natural infested pasture was performed, and the height of the grass was measured. Five peaks of engorged R. microplus females on animals and larvae infestations on pastures were observed. Being three and two peaks during the rainy and dry season of the year, respectively. Insolation (r = 0.8758; P = 0.00009; R²â€¯= 0.7670), rainfall (r = -0.8572; P = 0.0002; R²â€¯= 0.7348), maximum environmental temperature in summer (r = 0.9999; P < 0.0087; R²â€¯= 0.9988) and the saturation deficit of the atmosphere in autumn (r = -0.9789; P = 0.0211; R²â€¯= 0.9582) influenced the seasonal dynamics of R. microplus larvae on pastures. While the forage height did not influence the number of larvae found on pasture (r = 0.1545; P = 0.7682; R²â€¯= 0.0239). The comparison between the climatic data of the last 30 years with the current climatological data (2012 to 2019) in the state of Goiás, and the comparison with R. microplus population dynamics from other two locations in the same biome indicated that the increase of the environmental temperature due to the global warming possibly is a determining factor which increase the number of one or up two tick annual peaks. In addition, this climatic variable was responsible for increase the population density of ticks on pastures and animals observed in July/winter of the present study, in comparison to other regions with the same biome, as discussed in this work.

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.

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.

Identification of a non-host semiochemical from tick-resistant donkeys (Equus asinus) against Amblyomma sculptum ticks.

Amblyomma sculptum is a tick affecting animal and human health across Argentina, Bolivia, Paraguay and Brazil. Donkeys, Equus asinus, are known to be resistant to A. sculptum, suggesting that they can produce non-host tick semiochemicals (allomones), as already demonstrated for some other vertebrate host/pest interactions, whereas horses, Equus caballus, are considered as susceptible hosts. In this study, we tested the hypothesis that donkeys produce natural repellents against A. sculptum, by collecting sebum from donkeys and horses, collecting the odour from sebum extracts, and identifying donkey-specific volatile compounds by gas chromatography (GC) and coupled GC-mass spectrometry (GC-MS). From the complex collected blends, five main compounds were identified in both species. Hexanal, heptanal and (E)-2-decenal were found predominantly in donkey extracts, whilst ethyl octanoate and ethyl decanoate were found predominantly in horse extracts. One compound, (E)-2-octenal, was detected exclusively in donkey extracts. In Y-tube olfactometer bioassays 36 different A. sculptum nymphs were tested for each extract, compound and concentration. The dry sebum extracts and the compounds identified in both species induced neither attraction nor repellency. Only (E)-2-octenal, the donkey-specific compound, displayed repellency, with more nymphs preferring the arm containing the solvent control when the compound was presented in the test arm across four concentrations tested (p < 0.05, Chi-square test). A combination of a tick attractant (ammonia) and (E)-2-octenal at 0.25 M also resulted in preference for the control arm (p < 0.05, Chi-square test). The use of semiochemicals (allomones) identified from less-preferred hosts in tick management has been successful for repelling brown dog ticks, Rhipicephalus sanguineus sensu lato from dog hosts. These results indicate that (E)-2-octenal could be used similarly to interfere in tick host location and be developed for use in reducing A. sculptum numbers on animal and human hosts.

Assessment of primers designed for the subspecies-specific discrimination among Babesia canis canis, Babesia canis vogeli and Babesia canis rossi by PCR assay.

Canine babesiosis is an infectious disease caused by either Babesia gibsoni or Babesia canis protozoans. The latter is also classified under three different phylogenetic groups, referred to as subspecies B. canis canis, B. canis vogeli and B. canis rossi. The objective of the present study was to validate and standardize a PCR assay to discriminate the organisms at the subspecies level. First, the reference sequences of the 18S rRNA, 5.8S rRNA and 28S rRNA genes, including the internal transcribed spacer 1 (ITS1) and 2 (ITS2) of the most common species and subspecies of the genus Babesia were retrieved from the GenBank database. Subspecies-specific primers (BAB3, BAB4 and BAB5) and one genus-specific primer were designed from the alignment of the sequences. The PCR assays were evaluated in three different combinations of primer pairs in order to assure complete specificity for each reaction. The results of the tests had demonstrated effectiveness of the novel primer pairs BAB1/BAB3, BAB1/BAB4 and BAB1/BAB5 for the amplification of the subspecies-specific target fragments of 746 bp (B. c. canis), 546 bp (B. c. vogeli) and 342 bp (B. c. rossi) by PCR. The original enzymatic amplification assays with novel primers reported in this paper were confirmed to be a reliable tool for the specific discrimination among B. canis subspecies by single-step PCR assays.

Persistence and efficacy of a new formulation based on dog allomonal repellents against Rhipicephalus sanguineus sensu lato tick.

Beagles are less susceptible to Rhipicephalus sanguineus sensu lato tick due to the production of the allomones benzaldehyde and 2-hexanone. Our previous published work showed that these compounds can reduce tick burden on susceptible dogs. Here we tested the hypothesis that an increase in repellent dose and release rate could increase repellent efficacy and persistence. Slow-release formulations of these compounds, with higher doses and release rates, were tested on artificially-infested dogs. Ten dogs were randomly assigned to two groups with five dogs each. The treated group received collars with slow-release formulations of the compounds attached, while the control group received collars with clean formulations attached. Five environmental infestations were performed, with the number of ticks (at all stages) on the dogs being counted once a day for 40 days. No significant increase in repellent efficacy was observed with the higher doses and release rates, whereas a greater persistence in repellent activity was observed. Treatment with the formulations resulted in a two-to-three-fold reduction in the number of immature stage ticks for up to three weeks. However, the number of adults was similar in both groups. Loss of repellent activity after the third week of testing coincided with a marked change in the relative release rates for the two compounds. It is hypothesized that relative amounts, rather than absolute amounts, of repellent release from slow-release formulations are important for repellent activity. We also hypothesize that the avoidance of less-preferred hosts by ticks relies on olfactory-mediated perception of specific blends of volatile cues from less preferred hosts.

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.

In vitro repellency of DEET and ß-citronellol against the ticks Rhipicephalus sanguineus sensu lato and Amblyomma sculptum.

Rhipicephalus sanguineus sensu lato and Amblyomma sculptum can parasite humans and domestic animals and are vectors of pathogens, including zoonoses. Repellents are an important tool of tick control. This study aimed to compare the efficacy of N,N-diethyl- 3-methylbenzamide (DEET), a standard repellent, versus ß-citronellol in a Petri dish bioassay. A semicircle of filter paper (31.8cm2) was treated with 87µl of one of four concentrations (0.200, 0.100, 0.050 and 0.025mg/cm2) of ß-citronellol, DEET or solvent (ethanol). A head-to-head test was developed treating one side with increasing concentrations of ß-citronellol as above mentioned, against the highest concentration of DEET. Besides that a blank assay was performed. Three males and three females were placed in the middle of the plate and their location was evaluated 5, 10 and 30min after the test was initiated. As a result, the time had no significant effect on repellency response of the ticks exposed to both compounds and their concentrations. The repellency response raised according with the increase of concentration. Additionally, our findings indicate that the tick A. sculptum was more sensitive to the compounds tested and ß-citronellol showed a higher efficacy than DEET. In addition, ß-citronellol could be formulated to protect humans and other animals from R. sanguineus s. l. and A. sculptum infestation, as well as the diseases transmitted by these species.

Brown dog tick, Rhipicephalus sanguineus sensu lato, infestation of susceptible dog hosts is reduced by slow release of semiochemicals from a less susceptible host.

Domestic dog breeds are hosts for the brown dog tick, Rhipicephalus sanguineus sensu lato, but infestation levels vary among breeds. Beagles are less susceptible to tick infestations than English cocker spaniels due to enhanced production of 2-hexanone and benzaldehyde that act as volatile tick repellents. We report the use of prototype slow-release formulations of these compounds to reduce the burden of R. sanguineus s. l. on English cocker spaniel dogs. Twelve dogs were randomly assigned to two groups with six dogs each. The treated group received collars with slow-release formulations of the compounds attached, while the control group received collars with clean formulations attached. Five environmental infestations were performed, with the number of ticks (at all stages) on the dogs being counted twice a day for 45days. The counts on the number of tick stages found per dog were individually fitted to linear mixed effects models with repeated measures and normal distribution for errors. The mean tick infestation in the treated group was significantly lower than in the control group. For larvae and nymphs, a decrease in tick infestation was observed at the fifth count, and for adults, lower average counts were observed in all counts. The compounds did not interfere with the distribution of the ticks on the body of the dogs, as a similar percentage of ticks was found on the anterior half of the dogs (54.5% for the control group and 56.2% for the treated group). The biological and reproductive parameters of the ticks were not affected by the repellents. This study highlights for the first time the potential use of a novel allomone (repellent)-based formulation for reduction of tick infestation on susceptible dogs.

Neuronal projections from the Haller's organ and palp sensilla to the synganglion of Amblyomma americanum§.

The present study was conducted to elucidate the neuronal pathways between peripheral olfactory and taste sensilla and the synganglion in an Ixodidae tick species. The tarsus of the front legs (olfactory nerves) and the fourth palpal segment (gustatory nerves) of unfed Amblyomma americanum males and females were excised. A neuronal tracer, dextran tetramethylrhodamine, was used for filling of the sensory neurons. The synganglion preparations were examined using a confocal microscope. Neuronal arborizations from the Haller's organ were confined to the olfactory lobes and the first pedal ganglion. The estimated number of olfactory glomeruli ranged from 16 to 22 per olfactory lobe in the females. The number of glomeruli was not counted in males because they were densely packed. Sensory neurons associated with sensilla at the distal end of the palpal organ projected into the palpal ganglion in the synganglion through the palpal nerve. Gustatory sensory neurons associated with palpal sensilla projected into a commissure with several bulges, which are confined in the palpal ganglion. The findings of distinct projection patterns of sensory neurons associated with the Haller's organ and palpal organ in the lone star tick from this study advanced our knowledge on mechanisms of sensory information processing in ticks.

Quantification of brown dog tick repellents, 2-hexanone and benzaldehyde, and release from tick-resistant beagles, Canis lupus familiaris.

We have recently shown that repellency of the tick Rhipicephalus sanguineus sensu lato by the tick resistant dog breed, the beagle, is mediated by volatile organic compounds (VOCs) 2-hexanone and benzaldehyde present in beagle odour. Ectoparasite location of animal hosts is affected by variation in these odour components and their ratios. The aim of this study was to quantify the release rate, and the ratio, of 2-hexanone and benzaldehyde from beagles. The odour of three beagles was collected, for four days, over one week (day 0, day 1, day 4 and day 7). The compounds were identified using coupled high-resolution gas chromatography-mass spectrometry (GC-MS), and authentic standards of compounds were used to generate external calibration curves for quantification. Both compounds were found in all dogs on all days. The amount of benzaldehyde was always higher than that of 2-hexanone and so their ratio varied from unity, on average (over time) being 3.128±0.365, 1.902±0.390, 1.670±0.671ngmL(-1) for beagle 1, 2 and 3, respectively. There was no significant (p<0.05, F-test) effect of time. The overall mean was 2.233±0.387ngmL(-1). These results further previous findings by documenting the presence of 2-hexanone and benzaldehyde in beagle odour samples covering a 7-day period. This knowledge enables development of repellents to protect dogs from R. sanguineus s. l. infestation.

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.

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.