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Attract or repel Amblyomma sculptum ticks: Screening of semiochemicals
Veterinary Parasitology 278 (2020) 109036
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Research paper
Attract or repel Amblyomma sculptum ticks: Screening of semiochemicals a,
b
T
b
Lorena Lopes Ferreira *, Jaires Gomes de Oliveira Filho , Fernanda de Oliveira Silva , Ana Livia Lacerda Ferrazc, Gabriel Moura Mascarind a
Escola de Veterinária, Campus Pampulha, Avenida Antônio Carlos 6627, CP 567, CEP 31270-901, Belo Horizonte, Minas Gerais, Brazil Escola de Veterinária e Zootecnia, Universidade Federal de Goiás, Campus Samambaia, Avenida Esperança, s/n, Campus Universitário, CEP: 74690-900, Goiânia, Goiás, Brazil c Clarion Biociências Ltda, Rua 11, Qd 7. Lt 47 a 55, Polo Empresarial de Goiás, Aparecida de Goiânia, Goiás, Brazil d Empresa Brasileira de Pesquisa Agropecuária – Embrapa Meio Ambiente, Rodovia SP 340, km 127,5, CP 69, CEP: 13820-000, Jaguariúna, São Paulo, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Amblyomma cajennense sensu lato Attractant Field trial Repellent Alternative tick control Y-tube olfactometer
Amblyomma sculptum is a tick of medical-veterinary importance. Areas with high infestations need to be monitored, and parasitized hosts treated accordingly. Carbon dioxide (CO2) traps and acaricides are commonly deployed as control measures, although with some disadvantages such as high costs, challenging logistics and vertebrate intoxication. Semiochemicals have the potential to improve tick attraction to traps and monitoring devices and alleviate the burden of A. sculptum infestations. Four concentrations (10, 5, 2.5 and 1.25 %) of 13 semiochemical candidates (CO2 only at 5 % as the standard, benzaldehyde, benzoic acid, salicylic acid, 2,6 diclorophenol, R-limonene, S-limonene, methyl salicylate, 1-octen-3-ol, acetone, ammonium hydroxide, isobutyric acid and lactic acid) were tested on unfed A. sculptum nymphs and adults using a Y-tube olfactometer to evaluated repellence and attraction behaviors. All stages tested were attracted to CO2, whereas nymphs were repelled by benzaldehyde and R-limonene, both at 10 %, and isobutyric acid at 5 and 10 %. Nymphs were attracted by methyl salicylate, benzoic acid and salicylic acid, all at 1.25 %, and by ammonium hydroxide at 2.5 %. Males were attracted by benzoic acid at 2.5 %, while females were repelled by benzaldehyde at 5 %. Mixtures with the attractive compounds achieved no attraction response. The compounds that caused attractiveness in the olfactometer assay (CO2, methyl salicylate, benzoic acid, salicylic acid and ammonium hydroxide) were placed randomly in traps in a grassland plot naturally infested with A. sculptum in triplicate. Notably, dry ice (CO2) remained the best at luring ticks in the field (P < 0.001). Benzoic acid should be further investigated since attractant activity was strongly confirmed in both laboratory and field tests. On the other hand, isobutyric acid and R-limonene could be better exploited due to their repellent role revealed by the lab assay, which makes them worthwhile molecules as natural repellents for the management of this tick.
1. Introduction Ticks are globally distributed bloodsucking arthropods with ability to transmit a variety of pathogens of medical and veterinary importance to humans and other animals. They find their hosts and conspecifics by cues derived from semiochemicals that act as attractants or repellents. These stimuli (or odors) can come either from the host (kairomones and allomones) or from ticks of the same species (pheromones). Carbon dioxide (CO2) is known to be a universal attractant to hematophagous arthropods (Sonenshine and Roe, 2014). Amblyomma sculptum belongs to the Amblyomma cajennense complex (Nava et al., 2014), and parasitizes a huge variety of mammals,
particularly horses, capybaras, tapirs and humans. Of particular public health concern is that this tick is the main vector of Rickettsia rickettsii to humans in Brazil, which causes Brazilian Spotted Fever, a lethal disease (Labruna, 2009; Araujo et al., 2015; Sato et al., 2019). Tick parasitism of horses results in discomfort and anemia when infestation is high (Barros-Battesti et al., 2006). Areas where both tick infestation and spotted fever cases are detected need acarological surveillance, which is mostly done by deploying CO2 traps (Vieira et al., 2004; Pedro et al., 2016). The most current tool for controlling this tick still relies on spraying chemical acaricides. Nonetheless, disadvantages with CO2, like availability, market price and transport difficulties, coupled with resistance and environmental and animal toxicity caused by acaricides
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Corresponding author. E-mail addresses: [email protected] (L.L. Ferreira), jaires_fi[email protected] (J.G. de Oliveira Filho), [email protected] (F. de Oliveira Silva), [email protected] (A.L. Lacerda Ferraz), [email protected] (G.M. Mascarin). https://doi.org/10.1016/j.vetpar.2020.109036 Received 9 September 2019; Received in revised form 18 January 2020; Accepted 20 January 2020 0304-4017/ © 2020 Elsevier B.V. All rights reserved.
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by chance and tested. The mixtures tested were: ammonium hydroxide (2.5 %) + benzoic acid (1.25 %) + salicylic acid (1.25 %) + methyl salicylate (1.25 %), 1:1:1:1 v/v; benzoic acid (1.25 %) + methyl salicylate (1.25 %), 1:1 v/v and 1:2 v/v; ammonium hydroxide (2.5 %) + methyl salicylate (1.25 %), 1:1 v/v.
it is required the development of alternative control measures (Cançado et al., 2008; Sonenshine and Roe, 2014). Chemical ecology provides knowlodge and tools for managing pest arthropods since host-derived and arthropod-derived semiochemicals regulate the behavior of ticks, and thus it allows to design push-pull strategies for sustainable control interventions. Many compounds have been identified such as pheromones, kairomones and allomones for different tick species and have been used in attract-and-kill traps and slow release prototype of semiochemical-like collars (Sonenshine, 2004; Borges et al., 2007, 2015; Oliveira Filho et al., 2017; Ferreira et al., 2019). Compounds such as benzaldehyde, benzoic acid, salicylic acid, 2,6 dichlorophenol (2,6 DCP), limonene, methyl salicylate and 1-octen3-ol (octenol) have been considered as promising semiochemicals that could act on the behavior of A. sculptum (Gachoka et al., 2012; Soares and Borges, 2012). However, behavioral tests have yet to be performed to verify the influence of these substances on tick olfactory responses. New tools for controlling ticks are desirable, and semiochemicals are potential alternatives for managing A. sculptum. Thus, we performed olfactometer tests and field assays with A. sculptum ticks using semiochemicals in order to identify substances with attractive or repellent properties. These natural odorants could be used in the future to reduce the burden of this ectoparasite on livestock and humans.
2.4. Olfactometer assays Olfactometer assays were performed at Laboratório de Ecologia Química de Carrapatos, EVZ/UFG, Goiânia, Goiás, Brazil. The protocol from Ferreira et al. (2019) was followed. Briefly, an acrylic 31 cm long Y-tube olfactometer (each arm 13 cm, body 18 cm) with a removable cap was used for 50:50 distribution choice assays. Each arm was connected to a Büchner flask (50 mL, Pyrex®, USA) by a flexible polyvinyl chloride (PVC) hose (16 mm ø, Nalgene®, USA). A fluxometer (ParkerP3A, USA) with a charcoal filter was attached to the end of each Büchner flask and set at 0.1 L/min. Airflow was generated by a sprinkler (Dia-Pump®, Fanem, Brazil) connected to the opening of the olfactometer body by a PVC hose. The vacuum was kept outside of the test room to remove the gases. The olfactometer was positioned vertically for nymphs (Duijvendijk et al., 2017) and horizontally for adults (Carr et al., 2013). Immature stages occur during the cold and dry season while adults occur during warm and rainy months (Oliveira et al., 2003; Ramos et al., 2017), so a water jar was used to humidify the air system for adults, while only an odor jar was used for the nymphs. A smoke test was performed to evaluate the pattern of the odor plume and to determine whether the Y-tube olfactometer could be used successfully. Next, CO2 (positive control) was connected to one arm of the olfactometer and charcoal purified air to the other to evaluate tick preference. One piece (1 × 4 cm) of filter paper (Whatman qualitative, number 1) was treated with 11 μL of the test substance to compare against the solvent (ethanol or hexane). After treatment, the papers were dried in a fume hood (Permution, E. J. Krieger e Cia) for 1 min before use in the bioassay. The paper was transferred into the Büchner flask and the ticks were released individually in the olfactometer. Every 5 min, the arms were inverted and the olfactometer cleaned with ethanol (95 %) and new treated papers were used. A total of 36 unfed nymphs, males and females, were used for each substance and concentration, with each compound and concentration being tested in order. Each tick was observed for a maximum of 2 min to record its choice for the arm. A tick that did not choose either arm was tested again two more times; any tick that made no choice during the three attemps was discarded as it was considered inactive for unknown reasons (Ferreira et al., 2019). Repellence behavior occurs when the tick moves away from the odor source and attraction when the arthropod moves toward the odor source (Bissinger and Roe, 2014). The compound was considered a repellent when a significant number of ticks (P < 0.05) chose the control arm of the olfactometer, or an attractant when a significant number (P < 0.05) of ticks chose the arm containing the compounf of interest.
2. Material and methods 2.1. Ethical statement The use of animals was approved by the Committee on Ethical Animal Use of the Federal University of Goiás (CEUA/UFG), protocol number 024/2014. 2.2. Ticks Engorged females of A. sculptum were collected from naturally infested horses of Escola de Veterinária e Zootecnia of the Federal University of Goiás, Goiânia (EVZ/UFG), state of Goiás, Brazil, and from nearby regions. The tick colony was maintained as described by Ferreira et al. (2019). Larvae and nymphs were fed on Naïve rabbits (Oryctolagus cuniculus) using a feeding chamber glued on the rabbits’ back. Free living stages were kept in a climate chamber under total darkness (T = 27 ± 1 °C and RH ≥ 80 %, Eletrolab, São Paulo) inside plastic syringes with the top cut off and sealed with hydrophilic cotton. Unfed nymphs and adults aged 30–50 days post-ecdysis were used for the experiments. 2.3. Semiochemical candidates The in vitro tests investigated compounds that were previously identified from fed males of A. sculptum (Gachoka et al., 2012), electrophysiologically active to unfed males of the same species (Soares and Borges, 2012) and substances that reported to be attractive to hematophagous insects (Carr et al., 2013): CO2 5 %, benzaldehyde, benzoic acid, salicylic acid, 2,6 DCP, R-limonene, S-limonene, methyl salicylate, 2-nitrophenol, octenol, acetone, ammonium hydroxide, isobutyric acid and L - (+) lactic acid. Screening of semiochemicals involved testing them in increasing concentrations (1.25, 2.5, 5 and 10 %) to determine whether ticks respond or not to them in a dose-dependent manner. Pure ethanol or hexane served as a negative control and was used to dilute the substances tested, while 5 % CO2 was considered a positive control, since it is considered a universal attractant to bloodsucking arthropods. The CO2 was purchased from White Martins (Goiânia, Goiás, Brazil) while the other substances came from Sigma-Aldrich® (Germany). More details about manufacture code and purity of all substances used in tis study are provided in Supplementary Material, Table S1. Substances that attracted ticks in the olfactometer assay were mixed
2.5. Field assay The chemical candidates that caused attraction in the olfactometer test of nymphs and/or adults of A. sculptum −CO2 (dry ice), ammonium hydroxide, benzoic acid, salicylic acid and methyl salicylate — plus a blank control (no substance added, just an empty flask), were further tested in a field assay. The substances were purchased from SigmaAldrich® (Germany) and dry ice cubes from Dry Ice Gelo Seco (Goiânia, Goiás, Brazil). The experiment was conducted in a 2.3 km² grassland area, near a watercourse with capybaras, located at Fazenda Escola of the Federal University of Goiás, state of Goiás, Brazil. The surroundings were monitored by the epidemiological surveillance of the State due to a report of a case of Spotted Fever (Superintendência de Vigilância em 2
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Saúde (SUVISA, 2016). Two tests were conducted, from 10:00 to 13:00 (Brasília-DF time zone), on different days in October in the same area. Temperature and relative humidity on both days reached averages of 36 °C and 30 %, respectively. A preferred method adapted from Carr et al. (2013) was used to delineate this field trial. Cotton flannel sheets (60 × 60 cm) with double-sided tape on the edges were arranged to prevent ticks from escaping. A glass vial (4 cm high, 1 cm ø), containing 2 g of the pure test substance, was placed in the center of each flannel sheet. Pellets of dry ice were used to emanate CO2. A 30 min pilot test was initially conducted to verify tick infestation in the area. Three traps with 330 g of dry ice were placed in three distinct points of the vegetation and inspected after 30 min, with any captured ticks being released back into the environment. The test confirmed a natural tick infestation in the area. The pilot test also allowed the total time of CO2 sublimation to be determined, which was observed to be 2 h. The following day, the same area was sectioned into three sectors (Sector 1, Sector 2 and Sector 3) of 16.64 m² each spaced at 15 m apart along a straight line. Each sector received six cotton flannel sheets arranged in a rectangle and separated by 2 m. For the field trial, each sector received five glass vials with the pure compounds mentioned above, one empty glass as a negative control (blank) and 1 kg of dry ice as a positive control (Fig. 1). Glass vials with the tested substances tested were distributed randomly. After two hours (time of complete dry ice sublimation, as observed in the pilot study), the traps were carefully packed inside plastic bags, identified and taken to the laboratory for tick inspection and enumeration per life stage. At Laboratório de Ecologia Química de Carrapatos, EVZ/UFG, Goiânia, Goiás, Brazil, the ticks were removed from the double-sided tape and stored in falcon tubes with 70 % ethanol for later identification. A stereoscopic microscope (Leica mz9.5) was used to identify larvae to genus (Clifford and Anastos, 1960) and nymphs and adults to species, using Martins et al. (2010) and the identification key of Nava et al. (2014), respectively.
Fig. 2. Responses of Amblyomma sculptum nymphs 30–50 days post-ecdysis to different chemical substances in a Y-tube olfactometer assay. The positive control was CO2, while negative controls were hexane and ethanol. Asterisks indicate significant differences from a 50:50 distribution (binomial test: * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant). For each compound, 36 different nymphs were tested.
include in our model the effect accounting for within-positions between treatments in each block, as these treatments were arranged equidistantly from each other (2 m apart) as illustrated in Fig. 1; thus, there was no disparity in treatment distance that accounted for positional differences between treatments. The statistical model was implemented with the package “MASS” (Venables and Ripley, 2002) in R statistical environment (R Core Team, 2018). In both cases, a significance level of P < 0.05 was adopted.
3. Results 3.1. Olfactometer assay The negative controls, hexane or ethanol, did not cause attraction or repellency to A. sculptum nymphs and adults tested, as expected (Supplemmentary material: Tables S1, S2 and S3). The CO2 caused attraction to nymphs (χ² = 6.25, df = 1, P = 0.012), females (χ² = 4.694, df =1, P = 0.030) and males (χ² = 4.694, df =1, P = 0.030) of A. sculptum (Figs. 2 and 3). For the 13 candidates of repellents or attractants in the four concentrations tested, seven caused repellence or attraction behavior to A. sculptum nymphs, with three categorized as repellents (benzaldehyde, rlimonene and isobutyric acid) and four as attractants (methyl salicylate, benzoic acid, ammonium hydroxide and salicylic acid). To adults, only two compounds triggered some behavior. Benzaldehyde was repellent to females and benzoic acid attractive to males, whereas none of these test compounds was attractive or repellent for both sexes (Supplementary material: Tables S2, S3 and S4). In general, a higher concentration used of a certain substance was seen to be repellent, whereas the lowest concentration was scored as attractive.
2.6. Statistical analyses Data from the olfactometer assays were analyzed with the binomial distribution using a Chi-square test with Yates’ correction (Yates, 1934) to compare tick choices. For the field trial, the total tick count data were fitted to a generalized linear model with negative binomial distribution with log link function. In this model, the fixed effects were attributed to block (three sectors) and treatment (six test chemical compounds of interest). It is important to point out that we did not
Fig. 3. Response of Amblyomma sculptum adults 30–50 days post-ecdysis to different chemical substances in a Y-tube olfactometer assay. The positive control was blank (charcoal purified air), while negative controls were hexane and ethanol. Asterisks indicate significant differences from a 50:50 distribution (binomial test: * P < 0.05 and ns = not significant). For each compound, 36 different nymphs were tested. Legend: M (male), F (female).
Fig. 1. Schematic representation of the traps in the field assay. The flannels (60 × 60 cm) with the glass vials containing the test substances were randomly assigned within 2 m of each other in the grassland of the experimental area. Attractant candidates: CO2 (dry ice), ammonium hydroxide, benzoic acid, salicylic acid, methyl salicylate and a blank control. 3
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compounds did not capture any tick (Fig. 4). The CO2 traps showed the best performance in capturing ticks when compared to the other compounds tested (χ² = 11.69, df = 12, P < 0.001).
Repellency was observed to A. sculptum nymphs when isobutyric acid was tested at two higher concentrations (5 and 10 %) (χ² = 12.25, df = 1, P < 0.001; χ² = 30.25, df =1, P < 0.001). R-limonene (χ² = 6.25, df =1, P = 0.012) and benzaldehyde (χ² = 4.694, df =1, P = 0.030) acted as repellents at 10 % (Fig. 2). Females of A. sculptum were repelled by benzaldehyde at 5 % (χ² = 8.028, df = 1, P = 0.005) (Fig. 3). Salicylic acid (χ² = 4.694, df = 1, P = 0.030), benzoic acid (χ2 = 6.25, df = 1, P = 0.012) and methyl salicylate (χ² = 6.25, df =1, P = 0.012) were attractive at the lowest concentration (1.25 %) to A. sculptum nymphs. Ammonium hydroxide was attractive at 2.5 % (χ² = 6.25, df = 1, P = 0.012) (Fig. 2). Benzoic acid (χ2 = 6.25, df = 1, P = 0.012) at 2.5 % attracted only males (Fig. 2). Substances that caused significant attractiveness to A. sculptum nymphs in Y-olfactometer assays were mixed and tested. None of the mixtures elicited attraction or repellency to the nymphs. The mixtures were: ammonium hydroxide (2.5 %) + benzoic acid (1.25 %) + salicylic acid (1.25 %) + methyl salicylate (1.25 %), 1:1:1:1 v/v (χ² = 0.028, df = 1, P = 0.867); benzoic acid (1.25 %) + methyl salicylate (1.25 %), 1:1 v/v (χ² = 0.694, df =1, P = 0.404) and 1:2 v/v (χ² = 0.694, df =1, P = 0.404); ammonium hydroxide (2.5 %) + methyl salycilate (1.25 %), 1:1v/v (χ² = 0.028, df =1, P = 0.867).
4. Discussion The results from this study regarding the olfactory responses of A. sculptum are novel and provide insights into the chemical ecology response of this tick. Seven semiochemical candidates elicited a tick response in the olfactometer assay, while the field study (traps) did not reproduce these results. In general, higher concentrations of the tested substances evoked a repellent behavior, while lower concentrations induced an attractive behavior, in agreement with the literature (Takken and Knols, 2010; Bissinger and Roe, 2014). The results here indicate that the tick’s olfactory response is mediated by a dose-dependent manner. The tested substances will be discussed in the following sequence: compounds that repelled, compounds that attracted and compounds that did not cause a tick response. Benzaldehyde was a repellent to A. sculptum nymphs at 10 % and to females at 5 %, while isobutyric acid at the concentrations of 5 and 10 % repelled only nymphs. Both compounds have been previously identified in mammalian hosts (Steullet and Guerin, 1992, 1994; Borges et al., 2015; Jaleta et al., 2016). Benzaldehyde has been found to be good natural repellent of Rhipicephalus sanguineus sensu lato (Borges et al., 2015; Oliveira Filho et al., 2017). The present results for benzaldehyde differ from previous studies with A. variegatum (Apps et al., 1998; Lusby et al., 1991; Yunker et al., 1992). This divergence may be related to higher concentrations used in the olfactometer assays and due to differences in the chemical ecology of the evaluated species of Amblyomma. Isobutyric acid is a major component of triatomine alarm pheromone (Guerenstein and Lazzari, 2009), but was not found in fed males of A. sculptum (Gachoka et al., 2012). Limonene showed repellent activity toward A. sculptum nymphs. Marketed products, including shampoos and aerosol sprays, contain this substance with the aim of controlling arthropods of livestock and agriculture, such as fleas, ticks, mosquitoes, ants and bedbugs (Hink and Dufeey, 1990; Wilkins, 1999;
3.2. Field study A total of 93 ticks were trapped during the field trial comprising 43 larvae, 7 nymphs and 43 adults (21 females and 22 males). Larvae were identified belonging from the Amblyomma genus, nymphs Amblyomma cajennense sensu lato and adults A. sculptum. The traps with CO2 (positive control) captured 86.02 % of the ticks (80 ticks, Sector 1: 43 larvae, 4 nymphs, 3 females and 2 males; Sector 2: 8 males; Sector 3: 13 females and 7 males). While traps with benzoic acid captured 9.68 % of ticks (Sector 2: 2 nymphs, 1 female; Sector 3: 3 females, 3 males), followed by the negative control (flannel with an empty glass vial) that trapped only 3.23 % of the ticks (Sector 1: 1 nymph, 1 female and 1 male) and ammonium hydroxide 1.07 % (Sector 2: 1 male). The other
Fig. 4. Number of tick stages (larva, nymph, female and male) captured by traps in each sector during the field experiment. Legend: CO2: dry ice; B: blank; BA: benzoic acid; SA: salicylic acid; MS: methyl salicylate; AH: ammonium hydroxide. 4
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hydroxide, benzoic acid, salicylic acid and methyl salicylate, using a higher concentration to increase the release rate are necessary. We also suggest additional field assays that collect ticks in different seasons and at different altitudes and the use of distinct behavioral methods to verify attraction, since method performance depends on the tick behavior, vegetation, geographical origin and weather conditions, including changes in atmospheric pressure (Pellegrino et al., 2013; Dantas-Torres et al., 2013; Pedro et al., 2016). Benzoic acid was the only compound to attract unfed A. sculptum nymphs and adults in vitro and in the field, so this substance should be further investigated to confirm its role as a potential attractant to capture ticks in the environment during the non-parasitic stage and thus prevent host parasitism.
Pohlit et al., 2011). These results from these previous studies corroborate with the repellent activity found in the present work in regards to isobutyric acid and limonene,ince these compounds were able to control hematophagous arthropods. The CO2 displayed strong attraction and luring activities for males, females and nymphs of A. sculptum in both the olfactometer and field assays, which was expected since this volatile is recognized as a universal attractant for hematophagous arthropods (Takken and Knols, 2010). Nonetheless, methyl salicylate, ammonium hydroxide, salicylic acid and benzoic acid also showed attractiveness to A. sculptum nymphs in the olfactometer assay, but none of them attracted more ticks than CO2 in the field. It may be that the volatility of CO2 is responsible for trapping a higher number of ticks than the other substances tested. Methyl salicylate is a component of attraction-aggregation-attachment pheromones (Schoni et al., 1984; Diehl et al., 1991; Gachoka et al., 2012) and is considered a long-distance attractant (Norval et al., 1992), which could reasonably explain the present results. Methyl salicylate was recently confirmed to activate R. sanguineus s. l. in a semi-field assay (Carnohan et al., 2017). Ammonium hydroxide is an aqueous solution, 30 % of which is ammonia (NCBI, 2017) that can be found on the skin (Geier et al., 1999), in mammal breath (Norwood et al., 1992) and in tick excreta (Grenacher et al., 2001; Sonenshine et al., 2003), which indicates that these compounds could participate in tick chemoecology as observed in the our study. Benzoic acid and salicylic acid have been previously identified in a hexane extract of A. sculptum males (Gachoka et al., 2012). Benzoic acid is present in human breath (Phillips et al., 1999) and in bovine odor (skin and hair) evoking a questing response from Rhipicephalus microplus (Osterkamp et al., 1999). Salicylic acid, in turn, acts in the immune response of plants (Klessig et al., 2016). To the best of our knowledge, there has been no report of salicylic acid modulating tick behavior until the present study, which reinforces the need for more studies to verify the attractant potential for A. sculptum. The compounds 2,6 DCP, octenol, acetone and L-lactic acid did not elicit any behavior of the unfed stages of A. sculptum tested here. The compound 2,6 DCP is the most known ixodid sexual pheromone (Berger, 1972; Sonenshine, 1985; Borges et al., 2007; Louly et al., 2008). Both A. sculptum females and males produce 2,6 DCP, but the former produces lower concentrations than the latter. In addition, this compound can mediate part of the courtship (attract and mount) of fed individuals of this tick species (Louly et al., 2008; Gachoka et al., 2011, 2012). Feeding is a crucial event for metastriate ticks to become sexually mature and able to perceive a sexual pheromone (Sonenshine, 2004). So, the absence of response to 2,6 DCP is understandable since the ticks tested in the present study were not fed. Octenol and acetone have previously been described as eliciting responses in ixodid ticks (Sonenshine, 2006) and attracting hematophagous arthropods (McMahon et al., 2001; McMahon and Guerin, 2002; Bernier et al., 2006; Logan and Birkett, 2007). However, octenol is considered a species-specific compound, being attractant or repellent depending on the concentration and the hematophagous arthropod species (Xu et al., 2015). Using the single cell technique, Soares and Borges (2012) tested octenol in Haller’s organ sensilla of unfed A. sculptum and observed electrophysiological responses to concentrations below 10−6 M and 104 M, respectively; concentrations much lower than those used in the present study. Carr et al. (2013) observed acetone, at 5, 1 and 0.5 %, to be an attractant for Amblyomma americanum adults whereas L-latic acid to be repellent at 1%. All these discrepancies in tick behavioral responses between our findings and earlier studies could be attributed to the physiological conditions of ticks (parasitic or non-parasitic life cycle), instar level, species and range of concentration tested fot these substances (Sonenshine and Roe, 2014). For example, repellency is only observed at higher concentrations and volatilities that cause toxicity to the arthropod (Dautel, 2004; Takken and Knols, 2010; Bissinger and Roe, 2014). Despite these results, new field experiments with ammonium
5. Conclusion As expected, CO2 attracted all unfed stages of A. sculptum tested. Furthermore, some substances, such as ammonium hydroxide, benzoic acid, salicylic acid and methyl salicylate, demonstrated potential for attraction of A. sculptum nymphs. Conversely, isobutyric acid and Rlimonene exhibited repellent activity. All of these compounds should be further explored to verify performance in the field and perhaps be developed in traps for push-and-pull strategies, or used as active ingredient in repellent formulations. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments We thank the National Council of Technological and Scientific Development (CNPq), Brazil, for its financial support (Grant no. #/2012-7 and #/2014-0). Special thanks to Professor Lígia Miranda Ferreira Borges for the chemical ecology support and to provide the structure used in this work at Laboratório de Ecologia Química de Carrapatos, Centro de Parasitologia Veterinária, EVZ/UFG. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetpar.2020.109036. References Apps, P.J., Vlioen, H.W., Pretorrus, V., 1998. Aggregation pheromones of the bont tick Amblyomma hebraeum: identification of candidates for bioassay. J. Vet. Res. 55, 135–137. Araujo, R.P., Navarro, M.B.M.A., Cardoso, T.A.O., 2015. Spotted fever in Brazil: mortality study for epidemiological surveillance. Cad. Saúde Colet. 23, 354–361. Barros-Battesti, D.M., Arzua, M., Bechara, G.H., 2006. Carrapatos de importância médico veterinária da região neotropical: um guia ilustrado para identificação de espécies, primeira ed. Instituto Butantan, São Paulo. Berger, R.S., 1972. 2,6-Dichlorophenol, sex pheromone of the lone star tick. Science 177, 704–705. Bernier, U.R., Kline, D., Posey, K.H., 2006. Human emanations and related natural compounds that inhibit mosquito host-finding abilities. In: Frances, S., Strickman (Eds.), Insect Repellents: Principles, Methods, and Uses. CRC Press, pp. 77–100. Bissinger, B.W., Roe, R.M., 2014. Tick repellent research, methods, and development. In: 2a ed. In: Sonenshine, D.E., Roe, R.M. (Eds.), Biology of Ticks Vol 2. Oxford University Press, Oxford, pp. 383–408 2014. Borges, L.M.F., Ferreira, L.A.M., Silva, L.S., Oliveira, R.A., Mussi, S.V., Faria, K.A., Melo, L.S., Abud, L.J., Costa, G.L., Soares, S.F., 2007. Efficacy of 2,6-dichlorophenol lure to control Dermacentor nitens. Vet. Parasitol. 147, 155–160. Borges, L.M.F., Oliveira Filho, J.G., Ferreira, L.L., Louly, C.C.B., Pickett, J.A., Birkett, M.A., 2015. Identification of non-host semiochemicals for the brown dog tick, Rhipicephalus sanguineus sensu lato (Acari: Ixodidae), from tick-resistant beagles, Canis lupus familiaris. Ticks Tick Born. Dis. 6, 676–682. Cançado, P.H.D., Piranda, E.M., Mourão, G.M., Faccini, J.L.H., 2008. Spatial distribution and impact of cattle-raising on ticks in the Pantanal region of Brazil by using the CO2 tick trap. Parasitol. Res. 103, 371. Carnohan, L.P., Kaufman, P.E., Allan, S.A., Gezan, S.A., Weeks, N.I., 2017. Laboratory and
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Research paper
Attract or repel Amblyomma sculptum ticks: Screening of semiochemicals a,
b
T
b
Lorena Lopes Ferreira *, Jaires Gomes de Oliveira Filho , Fernanda de Oliveira Silva , Ana Livia Lacerda Ferrazc, Gabriel Moura Mascarind a
Escola de Veterinária, Campus Pampulha, Avenida Antônio Carlos 6627, CP 567, CEP 31270-901, Belo Horizonte, Minas Gerais, Brazil Escola de Veterinária e Zootecnia, Universidade Federal de Goiás, Campus Samambaia, Avenida Esperança, s/n, Campus Universitário, CEP: 74690-900, Goiânia, Goiás, Brazil c Clarion Biociências Ltda, Rua 11, Qd 7. Lt 47 a 55, Polo Empresarial de Goiás, Aparecida de Goiânia, Goiás, Brazil d Empresa Brasileira de Pesquisa Agropecuária – Embrapa Meio Ambiente, Rodovia SP 340, km 127,5, CP 69, CEP: 13820-000, Jaguariúna, São Paulo, Brazil b
A R T I C LE I N FO
A B S T R A C T
Keywords: Amblyomma cajennense sensu lato Attractant Field trial Repellent Alternative tick control Y-tube olfactometer
Amblyomma sculptum is a tick of medical-veterinary importance. Areas with high infestations need to be monitored, and parasitized hosts treated accordingly. Carbon dioxide (CO2) traps and acaricides are commonly deployed as control measures, although with some disadvantages such as high costs, challenging logistics and vertebrate intoxication. Semiochemicals have the potential to improve tick attraction to traps and monitoring devices and alleviate the burden of A. sculptum infestations. Four concentrations (10, 5, 2.5 and 1.25 %) of 13 semiochemical candidates (CO2 only at 5 % as the standard, benzaldehyde, benzoic acid, salicylic acid, 2,6 diclorophenol, R-limonene, S-limonene, methyl salicylate, 1-octen-3-ol, acetone, ammonium hydroxide, isobutyric acid and lactic acid) were tested on unfed A. sculptum nymphs and adults using a Y-tube olfactometer to evaluated repellence and attraction behaviors. All stages tested were attracted to CO2, whereas nymphs were repelled by benzaldehyde and R-limonene, both at 10 %, and isobutyric acid at 5 and 10 %. Nymphs were attracted by methyl salicylate, benzoic acid and salicylic acid, all at 1.25 %, and by ammonium hydroxide at 2.5 %. Males were attracted by benzoic acid at 2.5 %, while females were repelled by benzaldehyde at 5 %. Mixtures with the attractive compounds achieved no attraction response. The compounds that caused attractiveness in the olfactometer assay (CO2, methyl salicylate, benzoic acid, salicylic acid and ammonium hydroxide) were placed randomly in traps in a grassland plot naturally infested with A. sculptum in triplicate. Notably, dry ice (CO2) remained the best at luring ticks in the field (P < 0.001). Benzoic acid should be further investigated since attractant activity was strongly confirmed in both laboratory and field tests. On the other hand, isobutyric acid and R-limonene could be better exploited due to their repellent role revealed by the lab assay, which makes them worthwhile molecules as natural repellents for the management of this tick.
1. Introduction Ticks are globally distributed bloodsucking arthropods with ability to transmit a variety of pathogens of medical and veterinary importance to humans and other animals. They find their hosts and conspecifics by cues derived from semiochemicals that act as attractants or repellents. These stimuli (or odors) can come either from the host (kairomones and allomones) or from ticks of the same species (pheromones). Carbon dioxide (CO2) is known to be a universal attractant to hematophagous arthropods (Sonenshine and Roe, 2014). Amblyomma sculptum belongs to the Amblyomma cajennense complex (Nava et al., 2014), and parasitizes a huge variety of mammals,
particularly horses, capybaras, tapirs and humans. Of particular public health concern is that this tick is the main vector of Rickettsia rickettsii to humans in Brazil, which causes Brazilian Spotted Fever, a lethal disease (Labruna, 2009; Araujo et al., 2015; Sato et al., 2019). Tick parasitism of horses results in discomfort and anemia when infestation is high (Barros-Battesti et al., 2006). Areas where both tick infestation and spotted fever cases are detected need acarological surveillance, which is mostly done by deploying CO2 traps (Vieira et al., 2004; Pedro et al., 2016). The most current tool for controlling this tick still relies on spraying chemical acaricides. Nonetheless, disadvantages with CO2, like availability, market price and transport difficulties, coupled with resistance and environmental and animal toxicity caused by acaricides
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Corresponding author. E-mail addresses: [email protected] (L.L. Ferreira), jaires_fi[email protected] (J.G. de Oliveira Filho), [email protected] (F. de Oliveira Silva), [email protected] (A.L. Lacerda Ferraz), [email protected] (G.M. Mascarin). https://doi.org/10.1016/j.vetpar.2020.109036 Received 9 September 2019; Received in revised form 18 January 2020; Accepted 20 January 2020 0304-4017/ © 2020 Elsevier B.V. All rights reserved.
Veterinary Parasitology 278 (2020) 109036
L.L. Ferreira, et al.
by chance and tested. The mixtures tested were: ammonium hydroxide (2.5 %) + benzoic acid (1.25 %) + salicylic acid (1.25 %) + methyl salicylate (1.25 %), 1:1:1:1 v/v; benzoic acid (1.25 %) + methyl salicylate (1.25 %), 1:1 v/v and 1:2 v/v; ammonium hydroxide (2.5 %) + methyl salicylate (1.25 %), 1:1 v/v.
it is required the development of alternative control measures (Cançado et al., 2008; Sonenshine and Roe, 2014). Chemical ecology provides knowlodge and tools for managing pest arthropods since host-derived and arthropod-derived semiochemicals regulate the behavior of ticks, and thus it allows to design push-pull strategies for sustainable control interventions. Many compounds have been identified such as pheromones, kairomones and allomones for different tick species and have been used in attract-and-kill traps and slow release prototype of semiochemical-like collars (Sonenshine, 2004; Borges et al., 2007, 2015; Oliveira Filho et al., 2017; Ferreira et al., 2019). Compounds such as benzaldehyde, benzoic acid, salicylic acid, 2,6 dichlorophenol (2,6 DCP), limonene, methyl salicylate and 1-octen3-ol (octenol) have been considered as promising semiochemicals that could act on the behavior of A. sculptum (Gachoka et al., 2012; Soares and Borges, 2012). However, behavioral tests have yet to be performed to verify the influence of these substances on tick olfactory responses. New tools for controlling ticks are desirable, and semiochemicals are potential alternatives for managing A. sculptum. Thus, we performed olfactometer tests and field assays with A. sculptum ticks using semiochemicals in order to identify substances with attractive or repellent properties. These natural odorants could be used in the future to reduce the burden of this ectoparasite on livestock and humans.
2.4. Olfactometer assays Olfactometer assays were performed at Laboratório de Ecologia Química de Carrapatos, EVZ/UFG, Goiânia, Goiás, Brazil. The protocol from Ferreira et al. (2019) was followed. Briefly, an acrylic 31 cm long Y-tube olfactometer (each arm 13 cm, body 18 cm) with a removable cap was used for 50:50 distribution choice assays. Each arm was connected to a Büchner flask (50 mL, Pyrex®, USA) by a flexible polyvinyl chloride (PVC) hose (16 mm ø, Nalgene®, USA). A fluxometer (ParkerP3A, USA) with a charcoal filter was attached to the end of each Büchner flask and set at 0.1 L/min. Airflow was generated by a sprinkler (Dia-Pump®, Fanem, Brazil) connected to the opening of the olfactometer body by a PVC hose. The vacuum was kept outside of the test room to remove the gases. The olfactometer was positioned vertically for nymphs (Duijvendijk et al., 2017) and horizontally for adults (Carr et al., 2013). Immature stages occur during the cold and dry season while adults occur during warm and rainy months (Oliveira et al., 2003; Ramos et al., 2017), so a water jar was used to humidify the air system for adults, while only an odor jar was used for the nymphs. A smoke test was performed to evaluate the pattern of the odor plume and to determine whether the Y-tube olfactometer could be used successfully. Next, CO2 (positive control) was connected to one arm of the olfactometer and charcoal purified air to the other to evaluate tick preference. One piece (1 × 4 cm) of filter paper (Whatman qualitative, number 1) was treated with 11 μL of the test substance to compare against the solvent (ethanol or hexane). After treatment, the papers were dried in a fume hood (Permution, E. J. Krieger e Cia) for 1 min before use in the bioassay. The paper was transferred into the Büchner flask and the ticks were released individually in the olfactometer. Every 5 min, the arms were inverted and the olfactometer cleaned with ethanol (95 %) and new treated papers were used. A total of 36 unfed nymphs, males and females, were used for each substance and concentration, with each compound and concentration being tested in order. Each tick was observed for a maximum of 2 min to record its choice for the arm. A tick that did not choose either arm was tested again two more times; any tick that made no choice during the three attemps was discarded as it was considered inactive for unknown reasons (Ferreira et al., 2019). Repellence behavior occurs when the tick moves away from the odor source and attraction when the arthropod moves toward the odor source (Bissinger and Roe, 2014). The compound was considered a repellent when a significant number of ticks (P < 0.05) chose the control arm of the olfactometer, or an attractant when a significant number (P < 0.05) of ticks chose the arm containing the compounf of interest.
2. Material and methods 2.1. Ethical statement The use of animals was approved by the Committee on Ethical Animal Use of the Federal University of Goiás (CEUA/UFG), protocol number 024/2014. 2.2. Ticks Engorged females of A. sculptum were collected from naturally infested horses of Escola de Veterinária e Zootecnia of the Federal University of Goiás, Goiânia (EVZ/UFG), state of Goiás, Brazil, and from nearby regions. The tick colony was maintained as described by Ferreira et al. (2019). Larvae and nymphs were fed on Naïve rabbits (Oryctolagus cuniculus) using a feeding chamber glued on the rabbits’ back. Free living stages were kept in a climate chamber under total darkness (T = 27 ± 1 °C and RH ≥ 80 %, Eletrolab, São Paulo) inside plastic syringes with the top cut off and sealed with hydrophilic cotton. Unfed nymphs and adults aged 30–50 days post-ecdysis were used for the experiments. 2.3. Semiochemical candidates The in vitro tests investigated compounds that were previously identified from fed males of A. sculptum (Gachoka et al., 2012), electrophysiologically active to unfed males of the same species (Soares and Borges, 2012) and substances that reported to be attractive to hematophagous insects (Carr et al., 2013): CO2 5 %, benzaldehyde, benzoic acid, salicylic acid, 2,6 DCP, R-limonene, S-limonene, methyl salicylate, 2-nitrophenol, octenol, acetone, ammonium hydroxide, isobutyric acid and L - (+) lactic acid. Screening of semiochemicals involved testing them in increasing concentrations (1.25, 2.5, 5 and 10 %) to determine whether ticks respond or not to them in a dose-dependent manner. Pure ethanol or hexane served as a negative control and was used to dilute the substances tested, while 5 % CO2 was considered a positive control, since it is considered a universal attractant to bloodsucking arthropods. The CO2 was purchased from White Martins (Goiânia, Goiás, Brazil) while the other substances came from Sigma-Aldrich® (Germany). More details about manufacture code and purity of all substances used in tis study are provided in Supplementary Material, Table S1. Substances that attracted ticks in the olfactometer assay were mixed
2.5. Field assay The chemical candidates that caused attraction in the olfactometer test of nymphs and/or adults of A. sculptum −CO2 (dry ice), ammonium hydroxide, benzoic acid, salicylic acid and methyl salicylate — plus a blank control (no substance added, just an empty flask), were further tested in a field assay. The substances were purchased from SigmaAldrich® (Germany) and dry ice cubes from Dry Ice Gelo Seco (Goiânia, Goiás, Brazil). The experiment was conducted in a 2.3 km² grassland area, near a watercourse with capybaras, located at Fazenda Escola of the Federal University of Goiás, state of Goiás, Brazil. The surroundings were monitored by the epidemiological surveillance of the State due to a report of a case of Spotted Fever (Superintendência de Vigilância em 2
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Saúde (SUVISA, 2016). Two tests were conducted, from 10:00 to 13:00 (Brasília-DF time zone), on different days in October in the same area. Temperature and relative humidity on both days reached averages of 36 °C and 30 %, respectively. A preferred method adapted from Carr et al. (2013) was used to delineate this field trial. Cotton flannel sheets (60 × 60 cm) with double-sided tape on the edges were arranged to prevent ticks from escaping. A glass vial (4 cm high, 1 cm ø), containing 2 g of the pure test substance, was placed in the center of each flannel sheet. Pellets of dry ice were used to emanate CO2. A 30 min pilot test was initially conducted to verify tick infestation in the area. Three traps with 330 g of dry ice were placed in three distinct points of the vegetation and inspected after 30 min, with any captured ticks being released back into the environment. The test confirmed a natural tick infestation in the area. The pilot test also allowed the total time of CO2 sublimation to be determined, which was observed to be 2 h. The following day, the same area was sectioned into three sectors (Sector 1, Sector 2 and Sector 3) of 16.64 m² each spaced at 15 m apart along a straight line. Each sector received six cotton flannel sheets arranged in a rectangle and separated by 2 m. For the field trial, each sector received five glass vials with the pure compounds mentioned above, one empty glass as a negative control (blank) and 1 kg of dry ice as a positive control (Fig. 1). Glass vials with the tested substances tested were distributed randomly. After two hours (time of complete dry ice sublimation, as observed in the pilot study), the traps were carefully packed inside plastic bags, identified and taken to the laboratory for tick inspection and enumeration per life stage. At Laboratório de Ecologia Química de Carrapatos, EVZ/UFG, Goiânia, Goiás, Brazil, the ticks were removed from the double-sided tape and stored in falcon tubes with 70 % ethanol for later identification. A stereoscopic microscope (Leica mz9.5) was used to identify larvae to genus (Clifford and Anastos, 1960) and nymphs and adults to species, using Martins et al. (2010) and the identification key of Nava et al. (2014), respectively.
Fig. 2. Responses of Amblyomma sculptum nymphs 30–50 days post-ecdysis to different chemical substances in a Y-tube olfactometer assay. The positive control was CO2, while negative controls were hexane and ethanol. Asterisks indicate significant differences from a 50:50 distribution (binomial test: * P < 0.05, ** P < 0.01, *** P < 0.001, ns = not significant). For each compound, 36 different nymphs were tested.
include in our model the effect accounting for within-positions between treatments in each block, as these treatments were arranged equidistantly from each other (2 m apart) as illustrated in Fig. 1; thus, there was no disparity in treatment distance that accounted for positional differences between treatments. The statistical model was implemented with the package “MASS” (Venables and Ripley, 2002) in R statistical environment (R Core Team, 2018). In both cases, a significance level of P < 0.05 was adopted.
3. Results 3.1. Olfactometer assay The negative controls, hexane or ethanol, did not cause attraction or repellency to A. sculptum nymphs and adults tested, as expected (Supplemmentary material: Tables S1, S2 and S3). The CO2 caused attraction to nymphs (χ² = 6.25, df = 1, P = 0.012), females (χ² = 4.694, df =1, P = 0.030) and males (χ² = 4.694, df =1, P = 0.030) of A. sculptum (Figs. 2 and 3). For the 13 candidates of repellents or attractants in the four concentrations tested, seven caused repellence or attraction behavior to A. sculptum nymphs, with three categorized as repellents (benzaldehyde, rlimonene and isobutyric acid) and four as attractants (methyl salicylate, benzoic acid, ammonium hydroxide and salicylic acid). To adults, only two compounds triggered some behavior. Benzaldehyde was repellent to females and benzoic acid attractive to males, whereas none of these test compounds was attractive or repellent for both sexes (Supplementary material: Tables S2, S3 and S4). In general, a higher concentration used of a certain substance was seen to be repellent, whereas the lowest concentration was scored as attractive.
2.6. Statistical analyses Data from the olfactometer assays were analyzed with the binomial distribution using a Chi-square test with Yates’ correction (Yates, 1934) to compare tick choices. For the field trial, the total tick count data were fitted to a generalized linear model with negative binomial distribution with log link function. In this model, the fixed effects were attributed to block (three sectors) and treatment (six test chemical compounds of interest). It is important to point out that we did not
Fig. 3. Response of Amblyomma sculptum adults 30–50 days post-ecdysis to different chemical substances in a Y-tube olfactometer assay. The positive control was blank (charcoal purified air), while negative controls were hexane and ethanol. Asterisks indicate significant differences from a 50:50 distribution (binomial test: * P < 0.05 and ns = not significant). For each compound, 36 different nymphs were tested. Legend: M (male), F (female).
Fig. 1. Schematic representation of the traps in the field assay. The flannels (60 × 60 cm) with the glass vials containing the test substances were randomly assigned within 2 m of each other in the grassland of the experimental area. Attractant candidates: CO2 (dry ice), ammonium hydroxide, benzoic acid, salicylic acid, methyl salicylate and a blank control. 3
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compounds did not capture any tick (Fig. 4). The CO2 traps showed the best performance in capturing ticks when compared to the other compounds tested (χ² = 11.69, df = 12, P < 0.001).
Repellency was observed to A. sculptum nymphs when isobutyric acid was tested at two higher concentrations (5 and 10 %) (χ² = 12.25, df = 1, P < 0.001; χ² = 30.25, df =1, P < 0.001). R-limonene (χ² = 6.25, df =1, P = 0.012) and benzaldehyde (χ² = 4.694, df =1, P = 0.030) acted as repellents at 10 % (Fig. 2). Females of A. sculptum were repelled by benzaldehyde at 5 % (χ² = 8.028, df = 1, P = 0.005) (Fig. 3). Salicylic acid (χ² = 4.694, df = 1, P = 0.030), benzoic acid (χ2 = 6.25, df = 1, P = 0.012) and methyl salicylate (χ² = 6.25, df =1, P = 0.012) were attractive at the lowest concentration (1.25 %) to A. sculptum nymphs. Ammonium hydroxide was attractive at 2.5 % (χ² = 6.25, df = 1, P = 0.012) (Fig. 2). Benzoic acid (χ2 = 6.25, df = 1, P = 0.012) at 2.5 % attracted only males (Fig. 2). Substances that caused significant attractiveness to A. sculptum nymphs in Y-olfactometer assays were mixed and tested. None of the mixtures elicited attraction or repellency to the nymphs. The mixtures were: ammonium hydroxide (2.5 %) + benzoic acid (1.25 %) + salicylic acid (1.25 %) + methyl salicylate (1.25 %), 1:1:1:1 v/v (χ² = 0.028, df = 1, P = 0.867); benzoic acid (1.25 %) + methyl salicylate (1.25 %), 1:1 v/v (χ² = 0.694, df =1, P = 0.404) and 1:2 v/v (χ² = 0.694, df =1, P = 0.404); ammonium hydroxide (2.5 %) + methyl salycilate (1.25 %), 1:1v/v (χ² = 0.028, df =1, P = 0.867).
4. Discussion The results from this study regarding the olfactory responses of A. sculptum are novel and provide insights into the chemical ecology response of this tick. Seven semiochemical candidates elicited a tick response in the olfactometer assay, while the field study (traps) did not reproduce these results. In general, higher concentrations of the tested substances evoked a repellent behavior, while lower concentrations induced an attractive behavior, in agreement with the literature (Takken and Knols, 2010; Bissinger and Roe, 2014). The results here indicate that the tick’s olfactory response is mediated by a dose-dependent manner. The tested substances will be discussed in the following sequence: compounds that repelled, compounds that attracted and compounds that did not cause a tick response. Benzaldehyde was a repellent to A. sculptum nymphs at 10 % and to females at 5 %, while isobutyric acid at the concentrations of 5 and 10 % repelled only nymphs. Both compounds have been previously identified in mammalian hosts (Steullet and Guerin, 1992, 1994; Borges et al., 2015; Jaleta et al., 2016). Benzaldehyde has been found to be good natural repellent of Rhipicephalus sanguineus sensu lato (Borges et al., 2015; Oliveira Filho et al., 2017). The present results for benzaldehyde differ from previous studies with A. variegatum (Apps et al., 1998; Lusby et al., 1991; Yunker et al., 1992). This divergence may be related to higher concentrations used in the olfactometer assays and due to differences in the chemical ecology of the evaluated species of Amblyomma. Isobutyric acid is a major component of triatomine alarm pheromone (Guerenstein and Lazzari, 2009), but was not found in fed males of A. sculptum (Gachoka et al., 2012). Limonene showed repellent activity toward A. sculptum nymphs. Marketed products, including shampoos and aerosol sprays, contain this substance with the aim of controlling arthropods of livestock and agriculture, such as fleas, ticks, mosquitoes, ants and bedbugs (Hink and Dufeey, 1990; Wilkins, 1999;
3.2. Field study A total of 93 ticks were trapped during the field trial comprising 43 larvae, 7 nymphs and 43 adults (21 females and 22 males). Larvae were identified belonging from the Amblyomma genus, nymphs Amblyomma cajennense sensu lato and adults A. sculptum. The traps with CO2 (positive control) captured 86.02 % of the ticks (80 ticks, Sector 1: 43 larvae, 4 nymphs, 3 females and 2 males; Sector 2: 8 males; Sector 3: 13 females and 7 males). While traps with benzoic acid captured 9.68 % of ticks (Sector 2: 2 nymphs, 1 female; Sector 3: 3 females, 3 males), followed by the negative control (flannel with an empty glass vial) that trapped only 3.23 % of the ticks (Sector 1: 1 nymph, 1 female and 1 male) and ammonium hydroxide 1.07 % (Sector 2: 1 male). The other
Fig. 4. Number of tick stages (larva, nymph, female and male) captured by traps in each sector during the field experiment. Legend: CO2: dry ice; B: blank; BA: benzoic acid; SA: salicylic acid; MS: methyl salicylate; AH: ammonium hydroxide. 4
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hydroxide, benzoic acid, salicylic acid and methyl salicylate, using a higher concentration to increase the release rate are necessary. We also suggest additional field assays that collect ticks in different seasons and at different altitudes and the use of distinct behavioral methods to verify attraction, since method performance depends on the tick behavior, vegetation, geographical origin and weather conditions, including changes in atmospheric pressure (Pellegrino et al., 2013; Dantas-Torres et al., 2013; Pedro et al., 2016). Benzoic acid was the only compound to attract unfed A. sculptum nymphs and adults in vitro and in the field, so this substance should be further investigated to confirm its role as a potential attractant to capture ticks in the environment during the non-parasitic stage and thus prevent host parasitism.
Pohlit et al., 2011). These results from these previous studies corroborate with the repellent activity found in the present work in regards to isobutyric acid and limonene,ince these compounds were able to control hematophagous arthropods. The CO2 displayed strong attraction and luring activities for males, females and nymphs of A. sculptum in both the olfactometer and field assays, which was expected since this volatile is recognized as a universal attractant for hematophagous arthropods (Takken and Knols, 2010). Nonetheless, methyl salicylate, ammonium hydroxide, salicylic acid and benzoic acid also showed attractiveness to A. sculptum nymphs in the olfactometer assay, but none of them attracted more ticks than CO2 in the field. It may be that the volatility of CO2 is responsible for trapping a higher number of ticks than the other substances tested. Methyl salicylate is a component of attraction-aggregation-attachment pheromones (Schoni et al., 1984; Diehl et al., 1991; Gachoka et al., 2012) and is considered a long-distance attractant (Norval et al., 1992), which could reasonably explain the present results. Methyl salicylate was recently confirmed to activate R. sanguineus s. l. in a semi-field assay (Carnohan et al., 2017). Ammonium hydroxide is an aqueous solution, 30 % of which is ammonia (NCBI, 2017) that can be found on the skin (Geier et al., 1999), in mammal breath (Norwood et al., 1992) and in tick excreta (Grenacher et al., 2001; Sonenshine et al., 2003), which indicates that these compounds could participate in tick chemoecology as observed in the our study. Benzoic acid and salicylic acid have been previously identified in a hexane extract of A. sculptum males (Gachoka et al., 2012). Benzoic acid is present in human breath (Phillips et al., 1999) and in bovine odor (skin and hair) evoking a questing response from Rhipicephalus microplus (Osterkamp et al., 1999). Salicylic acid, in turn, acts in the immune response of plants (Klessig et al., 2016). To the best of our knowledge, there has been no report of salicylic acid modulating tick behavior until the present study, which reinforces the need for more studies to verify the attractant potential for A. sculptum. The compounds 2,6 DCP, octenol, acetone and L-lactic acid did not elicit any behavior of the unfed stages of A. sculptum tested here. The compound 2,6 DCP is the most known ixodid sexual pheromone (Berger, 1972; Sonenshine, 1985; Borges et al., 2007; Louly et al., 2008). Both A. sculptum females and males produce 2,6 DCP, but the former produces lower concentrations than the latter. In addition, this compound can mediate part of the courtship (attract and mount) of fed individuals of this tick species (Louly et al., 2008; Gachoka et al., 2011, 2012). Feeding is a crucial event for metastriate ticks to become sexually mature and able to perceive a sexual pheromone (Sonenshine, 2004). So, the absence of response to 2,6 DCP is understandable since the ticks tested in the present study were not fed. Octenol and acetone have previously been described as eliciting responses in ixodid ticks (Sonenshine, 2006) and attracting hematophagous arthropods (McMahon et al., 2001; McMahon and Guerin, 2002; Bernier et al., 2006; Logan and Birkett, 2007). However, octenol is considered a species-specific compound, being attractant or repellent depending on the concentration and the hematophagous arthropod species (Xu et al., 2015). Using the single cell technique, Soares and Borges (2012) tested octenol in Haller’s organ sensilla of unfed A. sculptum and observed electrophysiological responses to concentrations below 10−6 M and 104 M, respectively; concentrations much lower than those used in the present study. Carr et al. (2013) observed acetone, at 5, 1 and 0.5 %, to be an attractant for Amblyomma americanum adults whereas L-latic acid to be repellent at 1%. All these discrepancies in tick behavioral responses between our findings and earlier studies could be attributed to the physiological conditions of ticks (parasitic or non-parasitic life cycle), instar level, species and range of concentration tested fot these substances (Sonenshine and Roe, 2014). For example, repellency is only observed at higher concentrations and volatilities that cause toxicity to the arthropod (Dautel, 2004; Takken and Knols, 2010; Bissinger and Roe, 2014). Despite these results, new field experiments with ammonium
5. Conclusion As expected, CO2 attracted all unfed stages of A. sculptum tested. Furthermore, some substances, such as ammonium hydroxide, benzoic acid, salicylic acid and methyl salicylate, demonstrated potential for attraction of A. sculptum nymphs. Conversely, isobutyric acid and Rlimonene exhibited repellent activity. All of these compounds should be further explored to verify performance in the field and perhaps be developed in traps for push-and-pull strategies, or used as active ingredient in repellent formulations. Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgments We thank the National Council of Technological and Scientific Development (CNPq), Brazil, for its financial support (Grant no. #/2012-7 and #/2014-0). Special thanks to Professor Lígia Miranda Ferreira Borges for the chemical ecology support and to provide the structure used in this work at Laboratório de Ecologia Química de Carrapatos, Centro de Parasitologia Veterinária, EVZ/UFG. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.vetpar.2020.109036. References Apps, P.J., Vlioen, H.W., Pretorrus, V., 1998. Aggregation pheromones of the bont tick Amblyomma hebraeum: identification of candidates for bioassay. J. Vet. Res. 55, 135–137. Araujo, R.P., Navarro, M.B.M.A., Cardoso, T.A.O., 2015. Spotted fever in Brazil: mortality study for epidemiological surveillance. Cad. Saúde Colet. 23, 354–361. Barros-Battesti, D.M., Arzua, M., Bechara, G.H., 2006. Carrapatos de importância médico veterinária da região neotropical: um guia ilustrado para identificação de espécies, primeira ed. Instituto Butantan, São Paulo. Berger, R.S., 1972. 2,6-Dichlorophenol, sex pheromone of the lone star tick. Science 177, 704–705. Bernier, U.R., Kline, D., Posey, K.H., 2006. Human emanations and related natural compounds that inhibit mosquito host-finding abilities. In: Frances, S., Strickman (Eds.), Insect Repellents: Principles, Methods, and Uses. CRC Press, pp. 77–100. Bissinger, B.W., Roe, R.M., 2014. Tick repellent research, methods, and development. In: 2a ed. In: Sonenshine, D.E., Roe, R.M. (Eds.), Biology of Ticks Vol 2. Oxford University Press, Oxford, pp. 383–408 2014. Borges, L.M.F., Ferreira, L.A.M., Silva, L.S., Oliveira, R.A., Mussi, S.V., Faria, K.A., Melo, L.S., Abud, L.J., Costa, G.L., Soares, S.F., 2007. Efficacy of 2,6-dichlorophenol lure to control Dermacentor nitens. Vet. Parasitol. 147, 155–160. Borges, L.M.F., Oliveira Filho, J.G., Ferreira, L.L., Louly, C.C.B., Pickett, J.A., Birkett, M.A., 2015. Identification of non-host semiochemicals for the brown dog tick, Rhipicephalus sanguineus sensu lato (Acari: Ixodidae), from tick-resistant beagles, Canis lupus familiaris. Ticks Tick Born. Dis. 6, 676–682. Cançado, P.H.D., Piranda, E.M., Mourão, G.M., Faccini, J.L.H., 2008. Spatial distribution and impact of cattle-raising on ticks in the Pantanal region of Brazil by using the CO2 tick trap. Parasitol. Res. 103, 371. Carnohan, L.P., Kaufman, P.E., Allan, S.A., Gezan, S.A., Weeks, N.I., 2017. Laboratory and
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