Archives of Insect Biochemistry and Physiology 67:1–8 (2008) Decrease in DEET Repellency Caused by Nitric Oxide in Rhodnius prolixus Valeria Sfara,1* Eduardo N. Zerba,1,2 and Raúl A. Alzogaray1,2 N,N-diethyl-3-methylbenzamide (DEET) is widely used as an insect repellent; however, little is known about its mode of action. On the other hand, nitric oxide (NO) participates in the olfaction transduction pathway of insects. In this work, nitrosoacetyl-cysteine (SNAC), a nitric oxide donor, or dibutyril-cyclic-GMP (db-cGMP), the cyclic nucleotide analog, were applied on fifth instar nymphs of Rhodnius prolixus before exposing them to DEET, to obtain information about the possible role of NO/ cGMP system in the olfaction process. In the first place, we exposed the nymphs to several DEET concentrations (70, 700, 1,750, and 3,500 µg/cm2). All these concentrations produced a repellent effect. A decrease in repellency during the course of the experiment was observed when the nymphs were exposed to high concentrations of DEET (700 and 1,750 µg/cm2), suggesting an adaptation phenomenon. The pre-treatment of the insects with 15 µg /insect of SNAC or 2 µg/insect of dbcGMP produced a reduction of the repellency. An increase in locomotor activity was observed in insects exposed to 350 or 700 µg/cm2 DEET. Although exposure to 70 µg/cm2 DEET produced a high repellency response, it did not modify the insects’ locomotor activity. Insects treated with two doses of SNAC before being exposed to 350 µg/cm2 of DEET showed no differences in locomotor activity compared to controls. Arch. Insect Biochem. Physiol. 67:1–8, 2008. © 2007 Wiley-Liss, Inc. KEYWORDS: nitric oxide; olfaction; repellency; DEET; Rhodnius prolixus INTRODUCTION An insect repellent has been defined as “a chemical which causes insects to make orientated movements away from its source” (Dethier et al., 1960). Other authors state that an insect repellent is “a chemical or mixture of chemicals that, acting in the vapour phase, causes the insects to behave in ways which result in its movements away from the source of the material” (Barton Browne, 1977). N, N- diethyl-3-methylbenzamide (DEET) is the active ingredient of most insect repellent products in the market (Reeder et al., 2001). Its effectiveness has been shown against a number of haemato- phagous insects, including the triatomine Rhodnius prolixus (Buescher et al., 1985), the major vector of Chagas disease in Central America and north of South America (Schofield, 1994). DEET has also shown a repellent effect in Triatoma infestans, (Alzogaray et al., 2000; Sfara et al., 2006). In these studies, it was observed that pretreatment of T. infestans nymphs with the sulphydryl reagent N-ethylmaleimide (NEM), decreased the repellent effect of DEET (Alzogaray et al., 2000). The inhibition of DEET repellency in T. infestans by NEM pre-treatment was attributed to chemoreception blockage. An earlier work of our laboratory demonstrated the capacity of NEM to 1 Centro de Investigaciones de Plagas e Insecticidas (CIPEIN-CITEFA/CONICET), (B1603ALO) Villa Martelli, Prov. de Buenos Aires, Argentina 2 Universidad Nacional de General San Martín, (B1650CDL) San Martín, Prov. de Buenos Aires, Argentina Contract grant sponsor: Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET). Abbreviations used: db-cGMP = dibutiryl-cyclic-guanosine-monophosphate; DEET = N,N-diethyl-3-methylbenzamide; NO = nitric oxide; SNAC = nitroso-acetyl-cysteine; RC = repellency coefficient. *Correspondence to: Lic. Valeria Sfara, CIPEIN-CITEFA, JB de La Salle 4397, (B1603ALO) Villa Martelli, Prov. de Buenos Aires, Argentina. E-mail: firstname.lastname@example.org Received 13 November 2006; Accepted 19 May 2007 © 2007 Wiley-Liss, Inc. DOI: 10.1002/arch.20210 Published online in Wiley InterScience (www.interscience.wiley.com) 2 Sfara et al. interfere with T. infestans feeding and mating; this was also attributed to the blockage of chemoreception, particularly olfaction (Picollo et al., 1993). Klun et al. (2006) proved that DEET exerted an olfactory-based repellent effect on Aedes aegypti, Anopheles stephensi, and Phlebotomus papatasi. Nitric oxide (NO), an ubiquitous gaseous molecule present in most cell types, participates in the olfaction transduction pathway of insects (Bicker, 1998; Davies, 2000). The main function of this membrane-permeating molecule is the activation of soluble guanylyl cyclase, leading to the formation of cyclic GMP (cGMP) in target cells (Müller, 1997). Neurochemical investigations of NO/cGMP signalling in the nervous system of insects suggest it has critical functions in olfaction, vision, and mechanosensation (Bicker, 2001). Olfactory receptor cells respond to odorant stimulation with depolarization of the cell membrane. There is an underlying mechanism of transduction that converts the electrical signal into a chemical one, generally mediated by cyclic nucleotides such as cGMP, cAMP, and IP3 (Breer et al., 1992). S-nitrosothiols are nitric oxide donors that release NO, mimicking the effect of endogenous NO. During the last few years, special attention has been paid to S-nitrosothiols due to their important role in NO transport and storage in a wide range of physiological processes, including those mediated by the NO/cGMP system (Hogg, 2002; Ng and Kubes, 2003; Al-Saldoni and Ferro, 2004). The aim of this work was to obtain information about the possible role of the NO/cGMP system in the olfaction mechanisms underlying DEET repellency in R. prolixus. Chemicals N, N-diethyl-3-methylbenzamide (DEET) with 97% purity was purchased from Aldrich (Milwaukee, WI); acetone, analytical grade, was purchased from Merck (Darmstadt, Germany); db-cGMP was purchased from Sigma-Aldrich (Milwaukee, WI). Nitroso-acetyl-cysteine (SNAC) was synthesized in our laboratory by acid-catalyzed nitrosation of acetyl-L-cysteine as previously described (Mathews and Kerr, 1993). Briefly, 32.6 mg of N-acetyl- Lcysteine was weighed in a vial of 4 ml and dissolved in 250 µl of distilled water (Solution A). Similarly, 46 mg of NO2Na were dissolved in 500 µl of a 0.1% EDTA solution (Solution B). Then, 150 µl of Solution B was gently added to Solution A. These compounds react immediately, and the resulting solution becomes red. This red solution was acidified to pH = 2 by adding HCl 1N and then left to stand at room temperature for 5 min. The solution was then neutralized with NaOH 0.5 N and taken to a final volume of 5 ml with cold acetone, obtaining a 40 mM solution of SNAC in acetone. This solution was finally diluted 1:10 with acetone to obtain a 4-mM solution of SNAC. Both solutions were deaerated with argon. SNAC was prepared daily and the diluted stock solution was kept in the dark at –15°C until use. Synthesis by this method produces S-nitroso-N-acetylcysteine, that is >99% pure and stable at pH <3.0 (Byler et al., 1983). The exact final concentration of SNAC was determined using a spectrophotometer (Shimadzu UV-160) at 330 nm, based on the molar extinction coefficient of 727 (Mathews and Kerr, 1993). Recording Equipment MATERIALS AND METHODS Biological Material Fifth instar nymphs of Rhodnius prolixus, obtained from a susceptible strain kept in our laboratory since 1975, were used for bioassays. Insects were starved for a period of 15–25 days after emergence and kept in a temperature-controlled chamber at 28°C, under a 12:12 h (L:D) photoperiod. A closed-circuit black-and-white video camera (VC 1910, Sanyo Electrical Co., Tokio, Japan) was placed 15 cm above the centre of the test arena. An image analyzer (Videomex V, Columbus, OH) converted the analogue signal input from the video camera to digital data. Resolution was 256 × 192 pixels and the acquisition and processing speed was 30 frames s–1. In the monitor, the video signal colors are inverted and, therefore, white objects apArchives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. Nitric Oxide and Repellency in R. prolixus pear to be black and vice versa. Thus, the presence of insects in the arena was determined by a visual contrast between the individuals (white) and arena background (dark) and scored as the number of on pixels. Locomotor activity and repellency were recorded using Multiple Zones Motion Monitor for Videomex software, which records the movement of multiple objects in an area. Each set of data was imported and handled in a personal computer. Bioassays: Repellency Groups of four nymphs were used to measure repellency. The test arena floor was covered by circular pieces of Whatman No. 1 filter paper (Whatman International Ltd., Miadstone, England), 11 cm in diameter. Each piece of paper was cut into halves (Zone I and Zone II). Zone I was treated with 0.35 ml acetone and Zone II was treated with 0.35 ml of a DEET solution in acetone (70 µg/cm2). After acetone evaporation (30 min), both filter paper halves were fitted on the test arena floor. In order to determine the distribution of nymphs on the test arena, the TV field image was divided into two zones using Multiple Zones Motion Monitor for Videomex software. The area (expressed in pixels) occupied by nymphs in each zone during the experiment was recorded for 30 min. The results were expressed as Repellency Coefficient (RC) = A(I) – A(II) / A(I) + A(II), where A(I) is the area occupied by insects in Zone I and A(II) is the area occupied in Zone II. When RC = 0, the distribution of insects is random. RC values can vary between –1 (complete attraction) and 1 (complete repellency). The significance of the RC values were determined using one-way ANOVA and Tukey test for post hoc comparisons; 95% confidence limits were calculated as described by Sokal and Rohlf (1980). An RC was significantly different from 0 when this value is not included in the RC’s CL95%. An additional experiment was performed to determine the presence of a possible adaptation phenomenon to DEET. Groups of four nymphs were exposed to treated filter paper pieces with three concentrations of DEET (70, 700, and 1,750 µg/cm2) as previously described. The area occupied by the Archives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. 3 nymphs in each zone during the experiment was recorded during 20 min. To explore the existence of a possible adaptation phenomenon, the total experimental time was divided into two periods of 10 min each. Then the insects’ behavior during the first 10 min was compared to their behavior during the second period. Five independent replicates of each experiment were performed (N = 80). The RC was calculated for every concentration of DEET in each time interval, and then compared statistically using the Student’s t-test for dependent samples. Bioassays: Locomotor Activity Groups of four nymphs were used to measure locomotor activity.The test arena consisted of plastic Petri dishes, 9 cm in diameter, with a circular piece of filter paper covering the floor. Each filter paper was treated with 0.5 ml of different concentrations of DEET solutions in acetone (70, 350, and 700 µg/cm2). Filter papers treated with 0.5 ml acetone were used as controls. The Petri dishes were covered with gauze taped in the rear side of the dish, and then placed inside a glass ring (5 cm high and 10 cm diameter). Finally, the insects were placed on the gauze (the glass ring prevented the insects from escaping from the gauze). Locomotor activity was registered during 60 min with the video tracking technique described above. Four independent replicates of each experiment were performed (N = 64). The locomotor activity was expressed in units of pixels/area, where “pixels” indicates the number of pixels that turned from “on” to “off” and vice versa during the experimental time, and “area” indicates the number of pixels “on” (area occupied by the insects). The number of pixels that change their state when the insects walk depends on the number of total pixels “on”; due to changes in insect position, the number of total pixels “on” varies during the experimental time. For this reason, we used the unit of pixels/area to normalize the results (Alzogaray et al., 1997; Alzogaray and Zerba, 2001). The results were analyzed by means of one-way ANOVA and post hoc comparisons were made with Tukey’s test for unequal N. 4 Sfara et al. To determine the effect of SNAC on locomotor activity changes elicited by high concentrations of DEET, two SNAC solutions in acetone (1.5 or 15 µg/insect) were topically applied to nymph antennae (see below). Following this application, the treated insects were exposed to 350 µg/cm2 of DEET and the locomotor activity of pre-treated insects was measured during 60 min in the previously described device. Three independent replicates of each experiment were performed (N = 36). Results were compared with one-way ANOVA. Topical Treatment in Antenna Before Repellency Bioassays Both antennae of each insect were topically treated with solutions of SNAC in acetone (1.5 or 15 µg/insect). One µl per antenna of each solution was applied using a microsyringe provided with a dispenser (Hamilton Company, Reno, NV). This volume rapidly covers the complete antenna surface before the solvent evaporates. Four nymphs were used per treatment. An equal volume of acetone was applied to controls. In a previous experiment, it was demonstrated that the topical application of acetone (1 µl/antennae) had no any effect on the olfactory behavioral of the nymphs when exposed to DEET (data not shown). Immediately after the SNAC treatment, insects were placed on the respective test arena to measure the repellent response or locomotor activity elicited by DEET as described above. Three independent replicates of each experiment were performed (N = 36). The corresponding RCs were calculated, and then statistically compared using one-way ANOVA and Tukey’s test for post-hoc comparisons. Two concentrations of dibutiryl-cGMP (dbcGMP), a cGMP analogue, diluted in acetone, were tested (1 or 2 µg/insect). Each insect received 1 µl/antenna (both antennae were treated) of each db-cGMP solution via topical application, and four nymphs were used per treatment. An equal volume of acetone was applied to controls. Immediately after db-cGMP application, the insects were placed on the test arena with half its surface treated with 70 µg/cm2 of DEET, in order to determine repellency. Three independent replicates of each experiment were performed (N = 36). The RCs were calculated as previously described, and then statistically compared using one-way ANOVA and Tukey’s test for post-hoc comparisons. RESULTS Table 1 lists the RC values of untreated insects exposed to acetone alone or different concentrations of DEET (70, 700, 1,750, and 3,500 µg/cm2). The RC for the insects exposed to acetone alone was 0.028. This value was not significantly different from 0 (because 0 was included in its CL95%, P > 0.05), indicating that acetone did not affect the insect behavior and the distribution of the nymphs on the experimental arena was at random. All the concentrations of DEET produced a significant repellency (0 was not included in their respective CL95%, P < 0.05). Furthermore, no significant differences were observed among the effects of the concentrations of DEET tested (one way ANOVA, P < 0.05). The repellency produced by 70 µg/cm2 of DEET on untreated (RC = 0.70 ± 0.100) or acetone-treated (RC = 0.80 ± 0.086) insects did not differ significantly (P = 0.23, Student’s t-test for independent samples). Figure 1 shows the effect of insect pre-treatment with two doses of SNAC on the repellency elicited by 70 µg/cm2 of DEET. SNAC produced a dose-dependent decrease in the repellent effect of DEET: only the higher dose applied (15 µg/insect) reduced TABLE 1. Repellency Coefficients (RC) of Untreated R. prolixus Nymphs Exposed to Different Concentrations of DEET* DEET (µg/cm2) 0 (acetone control) 70 700 1,750 3,500 Repellency coefficient Standard error Confidence limits (95%) 0.028 a 0.70 b 0.59 b 0.73 b 0.68 b 0.0893 0.136 0.127 0.072 0.132 –0.147 to 0.203 0.433 to 0.966 0.341 to 0.839 0.589 to 0.871 0.421 to 0.939 *Acetone control consisted of the exposure of the insects to an experimental arena with both surfaces treated with acetone alone. Each RC value represents the mean of five independents replicates (N = 100). Different letters indicate significant differences (P < 0.01, one-way ANOVA, followed by Tukey test for unequal N for post-hoc comparisons). Archives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. Nitric Oxide and Repellency in R. prolixus Fig. 1. Repellency coefficients (RC) for 70 µg/cm2 of DEET on R. prolixus nymphs pretreated with nitrosoacetylcysteine (SNAC). Each bar represents the mean of three independent replicates (N = 36). Error bars are SEM. Different letters indicate significant differences (P < 0.05, one-way ANOVA). significantly the insect response to the repellent (Tukey test for post hoc comparisons, P < 0.05). In the other experiment, the insects were pretreated with db-cGMP. This compound reduced the insect response to DEET in a dose-dependent way (Fig. 2). Two doses of db-cGMP were tested, and only the higher one (2 µg/insect) produced a significant decrease of the repellency (Tukey test for post hoc comparisons, P < 0.05). Fig. 2. Repellency coefficients (RC) for 70 µg/cm2 of DEET on R. prolixus nymphs treated with dibutyryl-cyclic GMP (db-cGMP). Each bar represents the mean of three independent replicates (N = 36). Error bars are SEM. Different letters indicate significant differences (P < 0.05, oneway ANOVA). Archives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. 5 To determine whether the exposure to DEET produces an adaptation phenomenon, untreated nymphs were exposed to different concentrations of the repellent and its effect was determined in two consecutive time intervals of 10 min each. As shown in Figure 3, the effect of 70 µg/cm2 of DEET did not differ significantly between intervals (Student’s t-test for dependent samples, P > 0.05). However, when insects were exposed to 700 or 1,750 µg/cm2 of DEET, a significant decrease of the repellent effect was observed in the second interval compared with the first one (Student’s t-test for dependent samples, P < 0.05). The effect of several concentrations of DEET (70, 350, and 700 µg/cm2) on untreated insect locomotor activity is showed in Figure 4 (according to data in Table 1, all these concentrations produced repellency). The locomotor activity of nymphs exposed to the lowest concentration of DEET was not significantly different from the locomotor activity of the control group (Tukey test for post hoc comparisons, P > 0.05), but a significant hyperactivity was elicited by 350 and 700 µg/cm2 of the repellent (one-way ANOVA, P < 0.05). Finally, the effect of pre-treatment with SNAC on the hyperactivity elicited by 350 µg/cm2 of DEET Fig. 3. Repellency coefficients (RC) obtained in two time intervals, of untreated R. prolixus nymphs exposed to different concentrations of DEET. Each bar represents the mean of five independent replicates (N = 80). Error bars are SEM. Different letters indicate significant differences (Student’s t-test for dependent samples, P < 0.05). 6 Sfara et al. Fig. 4. Locomotor activity of untreated R. prolixus nymphs elicited by vapors of increasing concentrations of DEET. Each bar represents the mean of four independent replicates (N = 80). Error bars are SEM. Different letters indicate significant differences (P < 0.05, one-way ANOVA). was studied. Figure 5 shows that no significant effects were observed when the insects received 1.5 or 15 µg/antennae of SNAC (one-way ANOVA, P > 0.05). DISCUSSION The repellent effect of DEET has been widely described for several insect species. Most studies Fig. 5. Locomotor activity of R. prolixus nymphs treated with two concentrations of nitroso-acetylcysteine (SNAC) and exposed to DEET vapors (350 µg/cm2). Each bar represents the mean of three independent replicates (N = 36). Error bars are SEM. Equal letters indicate no significant differences among treatments (P > 0.05, one-way ANOVA). focus their attention on the repellency response of haematophagous insects, particularly mosquitoes. The effect of DEET on Triatomines has been described by our laboratory for T. infestans (Alzogaray et al., 2000; Sfara et al., 2006). These results show that DEET doses between 7 and 700 µg/cm2 are repellent for T. infestans nymphs. We have also observed that doses in the same range and higher were effective repellents for fifth instar nymphs of R. prolixus (Table 1). Many approaches have been made since the 1970s to understand the mode of action of insect repellents, particularly DEET. However, all the information available to the present is not enough to comprise a convincing model of the mode of action of this repellent. McIver (1981) suggested a model in which DEET molecules in the vapour phase reach the neuron membranes of chemosensory sensilla, where they interact with cell membrane lipids affecting the normal response of sensory neurons. Burton Browne (1977) considered it necessary to study certain topics related to orientation mechanisms involved in attraction, before discussing behavioural aspects of the repellency phenomenon. It has also been proposed that in Aedes aegypti mosquitoes (Dogan et al., 1999), DEET is an inhibitor of the attraction to lactic acid rather than a repellent in itself. However, this theoretical model is not strongly supported by experimental evidence, since it has been established that DEET itself does have a repellent effect. Our laboratory has shown the efficiency of DEET vapours in repelling triatomines (Alzogaray et al., 2000). We have also demonstrated that pre-treatment of T. infestans nymphs with a sulphydryl reagent, Nethyl maleimide (NEM), known to block chemoreception, produces a reduction in the repellency of insects exposed to DEET. Although it is accepted that DEET acts in the vapour phase, it is not clear whether there are specific receptors in the chemosensory sensilla of insects or not. Topical applications of a NO donor, SNAC, produced a significant decrease in the repellent response of R. prolixus nymphs when exposed to a repellent concentration of DEET. Many functions Archives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. Nitric Oxide and Repellency in R. prolixus of NO have been reported in the last few years. It has been demonstrated that the NO/cGMP system in the olfactory epithelium of rats is activated when odour stimuli are presented (Breer et al., 1992). A transient increase in cGMP and cAMP was observed when the epithelium was stimulated with odorants. In insects, NO participates as a retrograde messenger in sensory pathways of the visual system and in mechanosensory information processing (Bicker, 2001). It is also involved in the processing of chemosensory signals in the antennal lobes and in the transduction of primary olfactory signals (Müller, 1997). High doses of odorant cause a delayed but sustained increase in cGMP levels, suggesting its implication in mechanisms of olfactory adaptation as well as in sensory transduction following chemical stimuli (Murata et al., 2004; Nakamura et al., 2005). Treatment with a membrane-permeable cGMP analogue, db-cGMP, at a dose of 2 µg/insect, also produced a significant decrease in the repellent response of R. prolixus nymphs exposed to DEET. Considering DEET an odorant, we found that the NO/ cGMP cascade probably produced a decrease in the perception of the repellent, and thus, a reduction in the repellent response. To determine if this effect could be due to an olfactory adaptation phenomenon, groups of nymphs were exposed to high concentrations of DEET, in a preliminary experiment. When insects were exposed to 70 µg/cm2 of DEET, a normal repellent response was observed during the entire experimental time; however, when insects were exposed to concentrations of 700 and 1,750 µg/cm2, a significant decrease in repellent response was observed. Since the treatments with SNAC or db-cGMP was focused on the insect antennae, the results of this experiment suggest that the decrease in RC values can be due to a sensory adaptation phenomenon. However, the participation of some mechanism involving the central nervous system cannot be discarded and more experiments should be done to elucidate this question. Application of the membrane-permeable dbcGMP to the outer dendritic membrane of the moth’s olfactory receptor neuron reduced its reArchives of Insect Biochemistry and Physiology January 2008 doi: 10.1002/arch. 7 sponse to pheromones (Redkozubov, 2000). The observed attenuated response was attributed to the reduction of an elementary receptor current, which elicits nerve impulses and underlies the overall receptor current. It was suggested that cGMP, the formation of which appears to be initiated by the NO activation of soluble guanylyl cyclase (Bicker, 2001), is probably an adjustment factor of cell sensitivity in the olfactory system of insects, which could also be involved in olfactory adaptation processes (Redkuzubov, 2000). Exposure to increasing concentrations of DEET produced a significant increase in the locomotor activity of nymphs. This effect was observed when insects were continuously exposed to 350 and 700 µg/cm2 DEET during 60 min. Hyperactivity was not observed with the lowest dose of DEET tested (70 µg/cm2), although this dose did produce a significant repellent effect. The increase in locomotor activity was not reverted when insects were treated with SNAC before being exposed to 350 µg/cm2 DEET. This result suggests that hyperactivity produced by the exposure of R. prolixus to DEET could not involve the NO/cGMP transduction pathway; furthermore, the hyperactivity phenomenon could have a higher threshold than repellency. In summary, this study shows that the topical application of SNAC, a NO donor, and db-cGMP, a membrane-permeable analogue of cGMP, reduces DEET repellency in R. prolixus nymphs. 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