Sublethal effects of neuroactive compounds on pheromone response thresholds in male oriental fruit moths.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 1:331-344 (1984) Sublethal Effects of Neuroactive Compounds on Pheromone Response Thresholds in Male Oriental Fruit Moths C.E. Linn, Jr., and W.L. Roelofs Department of Entomology, New York State Agricultural Experiment Station, Genma The pheromone-mediated flight behavior of male Oriental fruit moths in a sustained-flight tunnel was observed after males were treated topically with sublethal concentrations of permethrin, carbaryl, chlordimeform, dieldrin, octopamine, serotonin, yohimbine, and cyproheptadine. With the exception of serotonin all compounds were found to disrupt one or more specific elements of the male precopulatory flight sequence. Among the insecticides, dieldrin was least active, whereas permethrin, carbaryl, and chlordimeform induced unique effects at specific phases of the sequence. Octopamine induced a hypersensitivity to the olfactory signal and mimicked one of the effects observed with chlordimeform. Yohimbine and cyproheptadine significantly decreased moth activation t o the chemical signal but did not alter flight performance in responding moths. Yohimbine and cyproheptadine also reversed the effects induced by octopamine. The results of our study show that the complex precopulatory sequence of behaviors exhibited by males is very sensitive to sublethal concentrations of a range of neuroactive compounds. Key words: sex pheromone, Oriental fruit moth, sublethal effects, permethrin, carbaryl, chlordimeforrn, dieldrin, octopamine, serotonin, yohimbine, cyproheptadine INTRODUCTION The sex pheromone-mediated flight behavior of male moths is a complex response that involves integration of visual and chemical inputs with a Acknowledgments: We gratefully acknowledge Kathy Poole, Marlene Campbell, and Laura Child for rearing the OFM. We thank Dr R.M. Hollingworth and Dr D. Soderlund for the generous gifts of test compounds, and also Dr Soderlund for his comments on the manuscript. We also thank Joe Ogrodnick, Rose McMillan-Sticht, and Bernadine Aldwinkle for the figures. This research was supported by National Science Foundation grant BNS 82-16752. Received February 27,1984; accepted April 13,1984. Address reprint requests to Dr C.E. Linn, Jr, Department of Entomology, NYS Agricultural Experiment Station, Geneva, NY 14456. 0 1984 Alan R. Liss, Inc. 332 Linn and Roelofs circadian rhythm that also regulates normal flight activity [l]. Females typically release a specific blend of compounds to which males respond optimally .In the case of the Oriental fruit moth,* Gvupholithu molesfa (Busck), the sex pheromone blend comprises three components: (Z)-8-dodecenyl acetate plus 6% (E)S-dodecenyl acetate and 3-30% (Z)-8-dodecenol [ 3 ] . We have been conducting studies using the OFM as a model organism to understand the factors that control or influence the response specificity of males to the chemical signal. One approach has been to analyze the behavior of males in a flight tunnel to a large number of blend-dosage combinations. In these tests male OFM responded in peak numbers to a narrow range of treatments around the blend and release rate produced by females. Analysis of the male response to suboptimal treatments showed evidence for several threshold effects on flight behavior, each associated with specific changes in blend or release rate [4-61. We report here a series of exploratory experiments to determine the efficacy of another approach to understanding how thresholds that control response specificity are regulated-that is, to observe the effects of sublethal concentrations of neuroactive compounds on male behavior. These compounds include insecticides with specific modes of action affecting various pathways in the nervous system, and neurotransmitters and neuromodulators that may affect the circadian rhythmicity of locomotory activity, as well as specific motor patterns. The results show that both the pheromonestimulated behavior and the thresholds for activation in male OFM can be dramatically altered with sublethal concentrations of a variety of compounds, and that different compounds influence male behavior in specific ways. METHODS Insects Male OF34 were reared on small green apples at 2 5 T , 16:8 L:D photoperiod. Adult males were segregated daily by age and kept under similar conditions in a room separated from females. Chemicals For all experiments the pheromone source was 10 pg of the optimal blend: Z8-12:Ac with 6% E8-12:Ac and 10% Z8-12:OH added . A stock solution of the three compounds (0.1 pglpl) was prepared in distilled Skelly €3 (principally hexanes). The ratio of compounds was checked by capillary GLC (45m Carbowax 20 M column). The stock solution (100 111) was applied to a rubber septum (red 5 x 9 mm, Arthur H. Thomas Co, Philadelphia), using a disposable capillary pipette. Male OFM were treated with the following compounds: the insecticides chlordimeform (as the free base), permethrin (5050 &:trans), carbaryl (ana- *Abbreviations: CNS = central nervous system; DMSO = dimethyl sulfoxide; E8-12Ac = (E)8-dodecenyl acetate; GLC = gas-liquid chromatography; OFM = Oriental fruit moth; 2812:Ac = (Z)-8-dodecenyl acetate; Z8-12:OH = (Z)-8-dodecenol. Neuroactive Compounds and Moth Pheromone Response 333 lytical standard), dieldrin (analytical standard); the biogenic amines DLoctopamine (Sigma Chemical Co, St. Louis, as the hydrochloride) and serotonin (5-hydroxytryptamine, as the hydrochloride, Sigma); and two known antagonists of octopamine-the alkaloid yohimbine (as the free base) and cyproheptadine. Chlordimeform, yohimbine, and cyproheptadine were gifts of Dr R.M. Hollingworth, Purdue University, and permethrin, carbaryl, and dieldrin were gfts of Dr D. Soderlund, NYS Agricultural Experiment Station. For each compound a dilution series was prepared and for all compounds except octopamine and serotonin, acetone was the solvent. For the latter two DMSO was utilized, as they are soluble in DMSO and we had hypothesized from previous studies [7Jthat this solvent might serve as a suitable carrier for the compounds across the cuticle. Procedure for Treating Insects Four-day-old males were treated 5 h prior to the normal period of testing in the flight tunnel (2 hr prior to initiation of scotophase). Males were first immobilized by placing them individually into 4-dram vials and then into a freezer ( - l 0 T ) for 10 min. Upon removal 1 p1 of solution was applied topically to the ventral thoracic region using a syringe. On each test day a series of concentrations were tested in addition to a control group receiving no treatment, one receiving the cold treatment alone, and one receiving cold exposure plus 1 pl of solvent. The solvent control group was treated first, followed by the series of concentrations in ascending order. Each compound was tested separately, with ten males treated per test day for each concentration and control. Prior to any treatment the syringe was cleaned and repeatedly rinsed with distilled acetone. Flight Tunnel Procedures The flight behavior of individual male OFM to the synthetic pheromone lure was observed in the flight tunnel of Miller and Roelofs . Treated and control groups were placed in the room housing the flight tunnel for 1h of acclimation prior to testing. For all tests the flight tunnel conditions were: 2 1 T , 350 lux, 50-70% RH, 35 cmlsec air velocity. The procedures and apparatus for handling and testing males were as previously described [5,6]. Males were scored for each behavior performed in the flight sequence : taking flight, stationary hovering flight near the release point, upwind anemotactic flight over a 1.5-m distance, landing at the source followed by a hairpencil display to the septum . For the present study a single extrusion of the hairpencils was scored as a display. Analysis was made of the number of males exhibiting each behavior based on the total number tested for all days (n = 70 for each concentration and control). For each treatment, behaviors were analyzed using the method of adjusted significance levels for proportions, P < 0.05 [lo]. For each compound control groups were also analyzed to v e d y that no significant effects had occurred in the cold- or solvent-treated insects. In the results to follow, all comparisons are between treated insects and untreated controls, as there were no significant effects observed between untreated, cold-treated, or solvent-treated controls in any 334 Linn and Roelofs of the tests. For the temporal data cited below the mean and standard deviation are indicated, based on the number of males exhibiting the behavior in question. RESULTS Mortality Evaluation of the percentage mortality for each compound (Fig. 1) was based on the number of males exhibiting no activity, even when prodded or touched, 24 h after treatment. For our experiments we arbitrarily defined a sublethal concentration as one resulting in <25% mortality 24 h after treatment. For the four insecticides tested, males were most sensitive to permethrin, followed by carbaryl, chlordimeform, and dieldrin. No lethal effects were observed for octopamine, serotonin, yohimbine, or cyproheptadine over the concentration range tested. Effects of Insecticides Significant effects on male behavior following exposure to sublethal concentrations were observed with all four insecticides tested. Treatment with permethrin resulted in a significant decrease in the number of males that oriented to the odor plume or initiated upwind flight (Fig. 2a). Following Fig. 1. Percentage mortality for each test compound recorded 24 h after treatment for a range of concentrations (pg) applied in 1 pl of solvent to male OFM. Clear area at the bottom of the figure shows the range of concentrations below 25% mortality and considered sublethal. Each value represents the mean and SD for 50 individuals tested. Neuroactive compounds and Moth Pheromone Response BEHAVIOR TF OR i c , UP CARBARYL I 335 HP 1 v) w 0: 150 b 130 DISTANCE FROM SOURCE (cm) Fig. 2. Flight response of male OFM to a synthetic sex pheromone source 5 h after topical application of permethrin (a), carbaryl (b), chlordimeform (c), dieldrin (d), octopamine (e), yohirnbine (f), and cyroheptadine (g). Behaviors are: taking flight (TF), plume orientation (OR), upwind flight (UP),and hairpencil display (HP) (at least one extrusion of the hairpencils). Values shown for 10 cm distance indicate males that landed on the 15 x 15 cm platform where source was located. N = 70 for each concentration. Dashed line indicates response of untreated controls. Within each behavior response values with different letters are significantly different, P < 0.05. Concentrations are pg applied in 1 PI of solvent to each moth; values in parentheses indicate percent mortality. 336 Linn and Roelofs BEHAVIOR TF UP OR HP 1 CHLORDIMEFORM C 1 . DISTANCE FROM SOURCE (cm) t 40 1 30 0 150 d 130 110 90 70 50 DISTANCE FROM SOURCE (cm) Figure 2 c and d. 10 Neuroactive Compounds and Moth Pheromone Response BEHAVIOR 1,F OR 337 UP 100 80 Lu 2 60 2 v) Lu p? 8 40 2a e BEHAVIOR TF OR $ 1 1 150 , , UP YOHlMBlNE I 130 I I I I 110 90 70 50 DISTANCE FROM SOURCE (cm) Figure 2 e and f. HP I I I I 30 10 0 Linn and Roelofs 338 flight initiation, males that did not orient to the plume typically flew a short distance and settled on the floor or side of the tunnel and became quiescent. Males that oriented to the plume but did not initiate upwind flight typically exhibited stationary flight for less than 3 s, thereafter settling on the floor of the tunnel. Males treated with 10P7toloP5 pg that flew to the source also took significantly longer to do so than did control insects (with pg flight initiation took 7.3 f 1.4s, n = 36, vs 1.1f 0.1 s, n = 64,and the time to fly upwind 1.5m took 11.3 4.8 s, n = 36, vs 3.1 k 1.1s, n = 64). Male response after treatment with carbaryl was not affected during the early orientation phase, but rather males were unable to initiate and sustain upwind flight (Fig. 2b). With loP5 to lo-' pg, for increasing percentages of males, the initial stage of the flight response was characterized by wide lateral excursions in the horizontal plane, often in excess of 40 cm (in control moths zigzag turns typically were 10-15 cm in lateral displacement across the center line of the plume). These excursions eventually resulted in the male losing contact with the plume and flying to the side of the tunnel. In addition, with to loP2 pg significant proportions of moths that initiated upwind flight failed to reach the source, leaving the plume space at varying distances from the source after exhibiting wide and apparently uncontrolled lateral casting patterns. Males that flew upwind to the source at loP5 pg took CY PROHEPTADINE UP BEHAVIOR TF OR -OK-0% loot HP _---__ ------- -- g,,, -------- - --------- I t 0 0 -0 --a 1 b _b Lu b b\- b b e-• b 10.' (0) b 100 (0) Lu oi 40 s 10' (0) 1 0 2 (0) I' 9 150 130 110 90 70 50 30 DISTANCE FROM SOURCE (cm) Figure 2 g. ;O 6 Neuroactive Compounds and Moth Pheromone Response 339 significantly longer than did control insects (15.7 f 4.9 s, n = 47, vs 2.8 f 1.1s, n = 65), with the males displaying stationary hovering flight several times along the flight path. Male response after treatment with chlordimeform was affected at all phases of the behavioral response (Fig. 2c). With 0.01 pg fewer males reached the source than initiated upwind flight, and the time taken to reach the source was 11.3 f 3.7 s (n = 41), vs 3.1 k 0.8 s (n = 63), for controls. In addition, the hairpencil display was significantly disrupted, with males exhibiting convulsive spasms upon reaching the platform and attempting to exert the hairpencil structures. In some cases these spasms lasted for up to 1 min (38.6 k 17.2 s, n = 41, for 0.01 pg), after which the male became quiescient and had to be removed from the platform. With 1pg chlordimeform, significantly fewer males oriented to the odor plume; those that did so spent significantly longer in this behavior than did controls (71.4 k 14.5 s, n = 37, vs 1.2 f 0.6 s, n = 66) and were unable to initiate upwind flight, instead flying vertically out of the odor plume. Male response was relatively unaffected by dieldrin (Fig. 2d) compared to permethrin, carbaryl, and chlordimeform. Male response appeared to be affected only in the early orientation phase, and no obvious adverse effects on flight performance (time to reach the source) or the hairpencil display were observed. Casual observations were also made of male activity during the period between treatment with insecticides and testing in the flight tunnel. These observations indicated that chlordimeform-treated males exhibited increased levels of spontaneous motor activity (walking andlor rapid bursts of wingfanning) and that the amount of activity appeared to be dose-related. The increased activity was observed within 30 min of application and was not observed with any of the other insecticides tested. Effects of Biogenic Amines Treatment with octopamine resulted in an increase in the number of males taking flight and orienting to the odor plume followed by a significant decrease in the number of males initiating upwind flight (Fig. 2e). Although the increase in male flight and plume orientation was not statistically significant, it was associated with a marked change in behavior exhibited by males upon being introduced into the tunnel. Males did not exhibit the typical wing-fanning andlor walking activation response; instead, upon being placed in the odor stream, males took flight immediately from a stationary position in the release cage. This apparent increase in sensitivity to the chemical signal was not reflected in increased levels of spontaneous activity prior to testing, as was observed with chlordimeform-treated males. Increasing the concentration of octopamine resulted not only in more males taking flight and orienting to the plume, but also significantly longer times spent in this phase of the response (47.2 k 16.2 s, n = 36, at 10 pg vs 1.1k 0.4 s, n = 61, for controls). Octopamine-treated insects that did fly upwind did not appear to be affected during flight, and completed this behavior in the same time as did controls (4.2 f 1.1s, n = 30, at 10 pg vs 3.6 k 1.3 s, n = 61). However, Linn and Roelofs 340 treated insects exhibited decreased levels of hairpencil displays at the source compared to controls. With 10 pg, males exhibited either no display or at most two displays, thereafter becoming quiescent on the platform. Control males typically exhibited several bouts of 3-5 displays covering a period of 30-60 S. In contrast to octopamine, serotonin (0.1-100 pg) did not significantly affect male behavior at any phase of the response, either in number of responding individuals or with respect to temporal factors. Effects of Antagonists Treatment of male OFM with yohimbine or cyproheptadine resulted in a significant decrease in the number of males initiating a flight response to the pheromone (Fig. 2 f,g). With the exception of two individuals, males that did not respond to the sex pheromone were capable of flight when touched with a blunt probe. In addition, no effects on flight performance were observed for males that completed the flight sequence, and there did not appear to be any effect on the duration or intensity of the hairpencil display. Yohimbine and cyproheptadine were also found to block the effect of octopamine on male behavior (Fig. 3). Application of 100 pg yohimbine or 1 pg cyproheptadine following 10 pg octopamine resulted in a reversal of the observed effects with each separate compound, and normal flight behavior was observed compared to control insects. DISCUSSION The results of our exploratory study indicate that the pheromone-mediated precopulatory flight and courtship behavior of male OFM is very sensitive to 100- 4 10' Octoparnine t 10'Cyproheptadine 0 ' 80 - 0 P 10' Octoparnine Lu v) 6 0 -----g_____--_____-_---___------_ g---d t lo2 Yohirnbine 60- v) Lu m2 2o t L I 150 I 130 1 1 I I 70 90 50 110 DISTANCE FROM SOURCE (cm) I I 1 30 10 0 Fig. 3. Flight response of male OFM to a synthetic sex pheromone source after topical application of octopamine followed by application of yohimbine or cyproheptadine. Symbols and analysis as in Figure 2. N = 70 for each concentration. Neuroactive Compounds and Moth Pheromone Response 341 a variety of neuroactive compounds. In general the insecticides we tested at sublethal concentrations did not affect the males’ ability to detect and respond to the signal, but they significantly disrupted the males’ ability to execute the oriented flight response upwind to the source. This disruption occurred in several ways, suggesting that different sites in the nervous system or levels of integration and motor control were being affected. We recognize, however, the difficulty in such extrapolations [ll], as our results do not provide direct evidence of central versus peripheral action of the compounds, their mode of entry, or their metabolic fate. The dominant effect observed in moths treated with permethrin was on the ability of males to sustain flight after detecting the pheromone signal. Permethrin is a synthetic pyrethroid, a group of compounds that block axonal conduction and transmission [El.A characteristic effect of these compounds is increased spontaneous bursting in neurons, leading eventually to suppression of activity in the neural pathway [E,13].The results of our study suggest that permethrin disrupted basic motor units involved in executing normal flight behavior. In another study  permethrin was found to decrease the activation response of male Pectinophoru gossypiellu (Saunders). The observed difference in results could be due to the difference in time between treatment and testing (5 h in the present study, 96 h in ), reflecting differing degrees of impact of the compound on the nervous system over time. In contrast to permethrin, carbaryl significantly disrupted the ability of males to execute the oriented upwind flight response. This is a more complex activity involving integration of visual and chemical imput with a guidance mechanism that influences the basic motor pattern controlling flight. Carbaryl is an inhibitor of acetylcholinesterase and thus is active in the CNS [El. Our results suggest that this compound affected CNS centers involved in integrating sensory stimuli and the guidance mechanism rather than specific motor pathways controlling flight coordination. The specific effects observed with permethrin and carbaryl contrast further with chlordimeform in which a number of concentration-dependent effects were observed. Increasing concentrations of chlordimeform disrupted upwind flight, the courtship display, plume orientation, and initiation of upwind flight. In addition, chlordimeform appeared to cause a concentrationrelated increase in spontaneous motor activity observed very soon after treatment and extending through the test period. This observation supports a number of other studies with insects, and lepidoptera specifically, showing that sublethal concentrations of chlordimeform induce in the early stages of toxicity a hyperactive state, effectively disrupting a number of behaviors [16,17J, including normal flight activity . No significant effects of behavior were observed with sublethal concentrations of the cyclodiene dieldrin. It is possible that this was due to an insufficient time for the material to exert an effect on the target tissues given the experimental protocol . The biogenic amines octopamine and serotonin were chosen for testing because of suspected roles in modulating CNS functions [19-261, and because direct effects of octopamine and serotonin on olfactory discrimination have been demonstrated in the honey bee, Apis melliferu . In the present study 342 Linn and Roelofs treatment with octopamine induced a marked increase in sensitivity to the pheromone signal, suggesting that the threshold for upwind flight had been shifted. We make this judgment based on the similarity of male response after treatment with octopamine to that observed to a higher than optimal concentration of the appropriate pheromone blend, where males typically take flight and remain in a stationary hovering flight near the release point for extended periods, thereafter leaving the odor plume by flying in a vertical pathway [5,23]. The absence of any observable effects in serotonin-treated insects does not eliminate this compound from consideration as a potential modulator of pheromone behavior. Serotonin has also been implicated in modulating circadian locomotor rhythms in the moth Agvotis ipsilon , in Peripluneta umericanu , and in Drosophilu melunogustev . Further testing with OFM at different times in the diurnal cycle might be necessary to observe an effect. It is also possible that topical applicaltion was not an effective means of introducing this compound into the hemolymph and neural tissues. A number of studies have demonstrated that chlordimeform interacts directly with octopaminergic receptor sites [19,27-331. In the present study octopamine and chlordimeform influenced male behavior in similar ways but also displayed distinct differences. Both compounds appeared to induce a state of hypersensitivity, but while that observed with octopamine was exclusive to the olfactory stimulus, chlordimeform induced a more general excitatory state [MI. The effect on plume orientation and initiation of upwind flight observed with all concentrations of octopamine was very similar to that observed with the 1pg concentration of chlordimeform. However, at other concentrations chlordimeform also disrupted the upwind flight performance of males and dramatically affected the hairpencil display, inducing spasms and loss of motor control. The hairpencil display was also disrupted by octopamine but in this case the effect was a lowering of the intensity of the display. If chlordimeform exerts its effects on the insect nervous system by interacting exclusively with octopaminergic sites, then the results suggest that chlorimeform had access to more of these sites than did octopamine. The observed sensitivity of octopamine-treated males to the chemical signal was effectively reversed by yohimbine and cyproheptadine, both known antagonists of one type of octopamineregic receptor [19,32]. In addition, yohimbine and cyproheptadine also exerted pronounced effects on male activation when presented alone, effectively eliminating male response to pheromone without significantly affecting the male’s ability to fly. These results, along with those from tests with chlordimeform, suggest that octopamine may be involved in modulating thresholds for pheromone-mediated behavior. However, further tests would be needed to confirm this, since it is known that chlordimeform, yohimbine, and cyproheptadine can interact at other sites [21,30,32]. The results of our study also have implications for two practical goals. First, it is clear that insecticides can exert significant sublethal effects on mating behavior and could thus have an important influence on pheromone field-trapping studies. It is also clear that sublethal effects of insecticides can contribute to reduced mating success in populations and thus contribute Neuroactive Compounds and Moth Pheromone Response 343 significantly to control of populations treated in conventional ways . Second, our flight tunnel assay allows for detailed analysis of a complex behavioral pattern that is critical for the organism’s survival. 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