Jmtrnal of Chemical Ecoh~gy, V~d. 21, No. 1I. 1995

SYNERGISM OF AN INSECT SEX PHEROMONE SPECIALIST NEURON: IMPLICATIONS FOR COMPONENT IDENTIFICATION AND RECEPTOR INTERACTIONS

M.S.

MAYER*

and R.E.

DOOLITTLE

htsect ,4ttractattts. Belutvior, and Basic Biology Rese~ir~7t L~tboratorv Agrieulntral Research Service, U.S. Det trtmettt f~'Agricultttre Gamesrille, ~7~n'i~hi32604

IReceived March 21. 1995; accepted July 13. 1995)

Abstract--Extracellular recordings show that the response to the sex pheromone component. (Z)-7-dodcccnyl acetate (Z7-12 : Ac) by the HS(a) antennal olfactory specialist neuron of the cabbage looper, Trichoplusia ni (H(ibnerl, is synergizcd by two synthetic sex pheromone analogs and tw~ components of the female sex pheromone. Complementary behavii~r,d measures of upwind flight and copulatory responses to mixtures of Z 7 - 1 2 : A t with the analogs and sex pheromone components in a wind tunnel produced behavior'a[ evidence consistent with the neuron's electrophysiological responses. The phenomenon of receptor neuron synergism impacts several areas and provides a note of caution for common practices of identifying secondary sex pheromone components only from field traps and/or wind tunnel tests. Key Words--Cabbage looper, responses, behavior.

electrophysiology, antenna, single cell

INTRODUCTION

C h e m i c a l c o m p o n e n t s o f f e m a l e m o t h s e x s i g n a l s are d e t e c t e d by o l f a c t o r y receptor neurons housed within characteristic, often sexually dimorphic, sensilla l o c a t e d o n the a n t e n n a . T h e s e r e c e p t o r n e u r o n s are s p e c i a l i z e d to d e t e c t l o w , p h y s i o l o g i c a l l y r e l e v a n t , a i r b o r n e c o n c e n t r a t i o n s o f p r i m a r i l y o n e o f the c o m p o u n d s that c o m p r i s e t h e f e m a l e s i g n a l . A t e l e v a t e d s t i m u l u s c o n c e n t r a t i o n s , however, even one of the most highly specialized neurons, the bombykol spe*To whom correspondence should be addressed, 1875 tX)98-0331/95/II(X)-1875507.50/0': 1995PlenumPublishingCt)rl~lnltitm

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MAYER AND DOOLtrTLE

cialist of Bombvx mori (L,). will respond to other compounds (Priesner, 1969). This is typical also of the HS(a) specialist of the cabbage looper moth, Trichoplusia ni (Hiibner), which detects the behaviorally most important component of its female chemical signal, (Z)-7-dodecenyl acetate ( Z 7 - 1 2 : A c ) (Mayer, 1993). As a consequence of this type of stimulus-strength-dependent specificity, it seems reasonable that the admixture of a sex pheromone component with a less active compound would elicit an additive response if both compounds are present at concentrations exceeding their individual thresholds. There are no reports of specialist neuron responses to mixtures of pheromone components or a particular component with other compounds at high stimulus intensities. At physiologically relevant airborne concentrations, Mayer {1993} showed that mixtures of pheromone components did not modify the response of the HS(a) specialist to Z 7 - 1 2 : Ac, but he did not assay these or other compounds at elevated stimulus intensities. What we examine in this report, inter alia, is whether or not a binary mixture stimulus, in which one of the components is Z 7 - 1 2 : A c , will elicit a mixed response lYom the HS(a) specialist neuron. This examination was prompted by our interest in the report of Burger et al. (1990) that 7-vinyldecyl acetate (7-VD) reduced trap captures of male false codling moths, Coptophtebia teucotrem (Meyrick), when incorporated into an otherwise effective sex pheromone bait. Comparison of the structure (Figure 1) of one of the two false codling moth sex pheromone components, (Z)-8-dodecenyl acetate (Z8-12 : Ac), with 7-VD shows that the length of the carbon chain between the double bond and the acetate function is the same in both molecules. In addition, the length of the carbon chain beyond the point of attachment of the double bond and the overall molecular size is the same. With these parameters in mind, we undertook the synthesis and behavioral and neurophysiologicat evaluation of molecules that would have a similar relationship to Z 7 - 1 2 : A c and, therefore, might be expected to stimulate the HS(a) neuron. We chose 6-vinyldecyI acetate (6-VD, Figure 1) and 10-vinyltetradecyl acetate (10-VT, Figure 1) as candidates. In the case of 6-VD, the relationship to Z 7 - 1 2 : A c is analogous to the relationship between 7-VD and Z 8 - 1 2 : A c of the false codling moth. Indeed, one can make the reasonable claim that because the vinyl group has no stereochemical restrictions, 7-VD can be spatially compared with the other false codling moth sex pheromone component, (E)-8-dodecenyl acetate (E8-12:Ac), as well. Inclusion of 10-VT in the study enabled the evaluation of the combined effect on the HS(a) neuron of Z 7 - 1 2 : A c and a molecule that had a longer carbon chain between the acetate function and the double bond than Z 7 - 1 2 : A c but retained the same four-carbon chain on the other side of the double bond. We tested 6-VD and 10-VT alone and in binary mixtures with Z7-12: Ac and found that these mixtures synergized both the response of the HS(a) neuron and upwind flight responses in a wind tunnel.

7', ni SYNERGISM

1877

7-VD

Z8-12:Ac

Z7-12 :Ac

6-VD O

10-VT 0

Z5-12:Ac

Z9-14:Ac 0 FIG. 1. Chemical structures of the compounds discussed in the text and/or used as odor stimuli in the electrophysiological and behavioral experiments: 7-vinyldecyl acetate, 7-VD; (Z)-8-dodecenyl acetate, Z8-12 :Ac; (Z)-7-dodecenyl acetate, Z7-12:Ac; 6-vinyldecyl acetate, 6-VD; 10-vinyltetradecyl acetate, 10-VT: (Z)-5-dodecenyl acetate, Z512 : Ac; and (Z)-9-tetvadecenyl acetate, Z9-14 :Ac.

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MAYER AND DOOI3TTLE

Until now, however, synergism has not been observed in moth sex pheromone specialist neurons. Synergism, in the sense that we use it for these studies, is the resultant response of the specialist neuron to the cooperative action of two or more discrete chemicals that is greater than the sum of the responses to the individual compounds. From this juncture, we gave thought to the possibility that synergism might be the means by which female volatile emission components that lack specialists are able to influence behavior. Two components that are considered to be part of the female sex signal were chosen for assay for the following reasons. One component, Z 9 - 1 4 : A c (Figure I). has been shown to be detected by a specialist neuron (Mayer and Mankin. 1990). although its effect on behavior is marginal ( L i n n e t al., 1984: Mayer and McLaughlin, 1991, 1993). The other component, Z5-12 : Ac (Figure I), was chosen because no specialist yet has been observed tbr its detection, but some preliminary assays in our wind tunnel at high stimulus intensities showed that it elicited some upwind flight behavior. Both of the compounds were examined alone and mixed with Z7-12 : Ac. The mixtures synergized both the neural response and upwind flight responses in a wind tunnel. One component, Z 5 - 1 2 : A c , synergized the response at near physiological airborne concentration levels, but Z 9 - 1 4 : A c did not. The implications of these results are discussed with respect to pheromone component identification, the effect of synergism on some aspects of behavior, and briefly, the potential use of synergists in field applications.

M E T H O D S AND M A T E R I A L S

h~sects. The moths were reared on a semisynthetic medium described in Guy et al. (1985). Males and females were separated during the pupal stage. After emergence, 40-60 males were maintained in 25 x 25 x 25-cm Plexiglas and fiberglass screen cages until assayed at 3-4 days of age. Two to four small plastic cups filled with a nutrient solution of 10% sugar-water on cotton were provided for each cage. The cages were maintained in cabinets at 25-27°C, 70-80% relative humidity on a 14:10 light-dark photoperiod. Chemicals. 6-VD and 10-VT were synthesized at this laboratory and were 98% pure by capillary column gas chromatographic (GC) analysis. Details of their synthesis will be the subject of a future report. The (Z)-7-dodecen-l-ol acetate (Z7-12:Ac) was a synthetic sample purified by HPLC and silicic acid column chromatographic procedures. It contained impurities totaling less than 0.1% of (E)-7-dodecen-l-ol acetate (E7-12 :Ac) or other compounds as determined by GC analysis. The sex pheromone components were analyzed by GC and were provided by R.R. Heath of this laboratory. Z 5 - 1 2 : A c was from Aldrich Chemical Co. (Milwaukee, Wisconsin) and was greater than 96% pure;

71. ni SYNERGISM

1879

Z 9 - 1 4 : Ac was from Sigma Chemical Co. (St. Louis, Missouri) and was 96% pure. Based on GC comparisons with authentic standards, none of the compounds contained significant amounts of high boiling point inclusions that could affect the emission rate. Electrophysiology. Action potentials were recorded from HS(a) specialists within sensilla by inserting electrolytically sharpened tungsten electrodes into the base of individual antennal sensilla (O'Connell, 1975; O'Connell et al,, 1983: Grant et al., 1989). The spikes were amplified by a Grass P-16 ACcoupled preamplifier and digitized, sorted, counted, verified, and stored digitally (Mankin et al., 1987), Pheromone Dispensers. All compounds and mixtures in both electrophysiological and behavioral assays were evaporated from calibrated glass dispensers (Mayer et al., 1987; Mayer, 1993). The dispensers were connected to two different glass devices that control the stimulus duration for the electrophysiological and behavioral assays. Chemical mixtures were released from the same dispenser. Slightly different airborne concentrations result from mixtures of 12and 14-carbon acetates because there is about a 10q'old evaporation rate difference (Mayer, 1993). The principles of the stirnu[us control device have been described by Grant et al. (1989) and Mayer (1993). The stimulus control device controls the slimulus and provides a constant 1200 ml/min of air to isolate the antenna from ambient laboratory air. A dosed dispenser is constantly purged with air at 200 ml/min. A bypass vacuum valve enables the stimulus to be injected countercurrently into the isolating airflow. Thus, a measured amount of pheromone from the dispenser is injected into the isolating airflow li)r a final airflow dilution of 1400 ml/min. The final airborne concentration can be calculated easily from the measured pheromone emission rate and the metered air flow. Control of the stimulus for behavioral assays is by a glass Y tube modified from that of Mayer and McLaughlin (1993) as described below. The Y tube was fitted with a glass diverter port that was fused at a point after the junction of the two female ground-glass joints and a longer nozzle. The diverter port was attached to a valve-controlled vacuum. One treated and one untreated dispenser, both purged constantly with air at 200 ml/min were inserted into the joints. During periods when the stimulus was off, the stimulus-laden air from the constantly purging dispensers was vented into the vacuum line. Upon activation of the valve, the stimulus was diverted into the center of the wind tunnel. To end the assay, the valve to the vacuum was opened, and the stimulus was vented into the vacuum. The final airflow dilution of the stimulus in this system was 400 ml/min. Wind Tunnel. Assays were conducted in a 183-cm-long, 60-cm-ID cylindrical Plexiglas tunnel. The input air was conditioned to 21-23°C, 50-70% relative humidity by an air handling unit (American Air Filter Inc., Louisville,

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MAYER AND DOOLITTLE

Kentucky) that brought in outside air, passed it through the laboratory's primary heating and cooling coils (96,000 BTU/hr and 129,000 BTU/hr capacity, respectively), filtered the conditioned air in turn through a 90% Varicel filter and then an Astrocel 95 % filter (capacity 1500 cfm), and passed the conditioned air into the tunnel. The airspeed in the tunnel was maintained at 15-20 cm/sec. The air in the tunnel was exhausted through the roof of the building. A white background with 37-ram-wide black strips 12 cm apart lay about I0 cm below the floor of the tunnel. Fiberglass sheeting diffused the light from an array of incandescent bulbs at the ceiling, controlled by a rheostat, to illuminate the tunnel uniformly. The light intensity at the center of the tunnel was maintained at 0.3 lux. The principle of this tunnel is the same as for other tunnels described by Baker and Linn (1984) and Miller and Roelofs (1978), except that it uses air drawn from the laboratory air supply. The plume produced from a dispenser loaded with tissue papers treated with NH4OH and HCI was "'turbulent" as defined by Mafra-Neto and Card6 (1994). Behavior. For behavioral assays individual male moths were released from a holding cage centered downwind from the stimulus source. The behaviors observed were typical of moths flying upwind to a pheromone source and were characterized by a combination of opportunistic optomotor-chemoanemotaxis and/or a program of sell-steered countertuming (Baker and Haynes, 1987). The final, most distinctive, and perhaps the most definitive of sex pheromone-stimulated intrinsic behaviors released by Z 7 - 1 2 : A c are those just prior to and during copulation. Typically, this behavior occurs at the stimulus source and is characterized by ventrad abdominal thrusts, extrusion of claspers, and the eversion of both the lateral and temlinal abdominal hairpencils. Relationship of Del)loyed Stimulus Intensities to a Female. As discussed in Mayer (t993~, it is necessary to reference the stimulus intensity to that of a female to both ensure the selectivity of the HS(a) specialist and maintain the environmental relevance of the stimulus. A female's instantaneous emission of Z7-12"Ac averages about 10 ng/min (Bjostad et al., 1980). More recent timeaveraged measures support an average emission rate of about 4 ng/min (Haynes and Hunt, 1990; Hunt et al., 1990~ Hunt and Haynes, 1990). On a logarithmic scale such a difference is within half an order of magnitude of our estimate. Each of the other components of the sex pheromone, (Z)-5-dodecenyl acetate (Z5-12 : Ac), (Z)-7-tetradeccnyl acetate (Z7-14 : Ac). (Z)-9-tetradecenyl acetate (Z9-14: Ac), dodecenyl acetate (12 : Ac), and AI l-dodecenyl acetate (1112: Ac), arc considered to reflect their ratios to Z7-12 :Ae as deternfincd by Bjostad et al. (1984) from time-averaged collections of airborne volatiles: 0,0935, 0.0105, 0.0046, 0.0719, and 0.0278, respectively, although some differences may exist (Haynes and Hunt, 1990; Dunkelb[um and Mazor, 1994). Thus, if 10 ng/min of Z 7 - 1 2 : A t were emitted into the dispenser used for the neurophysiological experiments, the airborne concentration at the antenna

T. ni SYNERGISM

1881

of each of these components due to dilution by 1400 cm3/min air (200 cm~/min from the dispenser + 1200 cm3/min from the stimulus delivery system) would average, as follows: Z 7 - 1 2 : A c , 3.2 x 10 -11 M; Z 5 - 1 2 : A c , 2,9 x 10 -12 M; Z 7 - 1 4 : A c , 2.9 x 10-~3 M ; Z 9 - 1 4 : A c , 1.3 x 10 ~3M; 12:Ac, 2.3 x 10 -~2 M; and A 11-12 : Ac, 8.8 x 10-13 M. Correspondingly, the airborne concentration of each of these components at the nozzle of the dispenser in the windtunnel experiments due to dilution by 400 cm'~/min air (200 cm3/min from one treated and one untreated dispenser) would be: Z7-12 :Ac, 1,1 × 10 -m M; Z5-12:Ac, 1.0 x 10 ~ M ; Z 7 - 1 4 : A c , 1.0 × 10 -I-~ M; Z 9 - 1 4 : A c , 4.5 x 10 -13 M; 12:Ac, 7.9 x 10 -t-" M, and A l l - 1 2 : A c , 3.1 x l0 -~-~ M. There may be further dilution by a factor of about 8 within the plume of the wind tunnel (Mayer and McLaughlin, 1991). Estimation of Airborne Stimulus Concentration. Details of the estimation of the airborne stimulus concentration of the odorants deployed in these studies are in Mayer et al., (1987) and Mayer (1993). In essence, the estimate of the airborne concentration of the various compounds derives from gas chromatographic, radiometric, and electrophysiological measures of the emission rate of Z 7 - 1 2 : A c t¥om the same glass dispensers used in these assays (Mayer et al., 1987). From the established Z 7 - 1 2 : Ac emission rate and additional gas chromatographic measures of emission rates at high dispenser dosages, Mayer (1993) estimated the airborne concentration of the other cabbage looper sex pheromone components by extrapolation. The emission rate of 6-VD was assumed to be the same as that tbr 14-carbon compounds. There are no measures for the emission rate of an 18-carbon compound from the dispenser used here, and consequently the emission rate tot 10-VT was judged to be about 10 times less than the emission rate measured of (Z)-I l-hexadecenal (Mayer et al., 1987).

RESULTS The single most behaviorally active sex pheromone component, Z7-12 :Ac, elicits reliable responses from the HS(a) specialist at airborne concentrations at or lower than about 2 x 10~3 M (Table 1) (Mayer, 1993). Among the eight HS(a) specialists assayed, this concentration of Z 7 - 1 2 : A c elicited an average of 8,7 _+ 2.6 (SEM) action potentials ( =spikes)/sec. Five other HS(a) specialists in the test series responded with an average of 40.7 + 10.9 spikes/sec to 2.1 x 10 ~ M Z 7 - 1 2 : A c . The same eight neurons that were exposed to Z 7 12 : Ac alone also responded reliably to 6-VD alone at an airborne concentration of - 1 X 10 - l ° M (Table 1). They did not respond reliably to 10-VT alone at either level tested. The response of the HS(a) specialists to admixtures of Z7-12 :Ac with either 6-VD or 10-VT was greater than the sum of the responses elicited by

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MAYER AND DOOLITTLE

TABLE I. RESPONSE OF

HS(a)

ANTENNAL SEX PHEROMONE SPECIALIST RECEPTOR

NEURONS TO INDICATED STIMULI

Airborne synergen concentration (M)" None 6-Vinyldecyl acetate

Average spikes/see HS(a) neurons" with airborne Z 7 - 1 2 : A c concentration (M) 0 0.3 + 0.1 (9) b a'

- 2 x 10 i_, 4.4 _+_ 3.9 (8)a - 1 x 10 I~ 0.6 + 0 . 3 ( 8 ) a - 2 x 10 ii 1.9 _+ 0.718)a - 1 x 10 m 23.6 + 8 . 9 ( 8 ) b 10-Vinyltetradecyl acetate - 3 x 10 i~ 3.7 + 2 . 8 ( 8 ) a - 3 x I0 ,2 6.0 + 2.6 (7)a

1.9 x 10 is 8.7 +_ 2.6 (8)a 17.1 35.1 42.7 73.5

2.1 z 10 -II 40.7 + 10.9 (5) d

+ 4.7 (8)a + 8.7(7)b + 13.6(6)b 5:37.1 (5)b

47.9 + 12.8(8)b 70.4 _+ 23.9 (4)b

"The emission rate and airborne stimulus concentration of 6-VD is estimated from the emission of Z I 1-16: AI because of their similarity in molecular weight: concentration of 10-VT stimulus is estimated as 10x less than the emission o f Z I 1-16:AI (14). t'Number of neurons measured. 'Different letters within a column indicate means that differ from the response m an untreated dispenser or 1.9 x 10 ~3 M Z 7 - 1 2 : A c alone at the P < 0.01 level of significance (Studem's t test). dMixtures of the synergens with 2.1 × 10 ~ M Z7-12 : Ac were not measured because the resultant responses were overdriven spike trains characterized by significantly decremented spikes lbllowcd by an abnormally elevated spontaneous activity.

either of the two stimuli alone, thus fulfilling the definition of synergism (Table 1; Figure 2). Moreover, the response was proportional to the logm M concentration of 6-VD added to constant levels of Z7-12 :Ac. The four values for 6-VD mixtures fit the regression equation, log spikes/see = 5.6 + 0.02 (log M) (r 2 -- 0.987; SE = 0.204; P < 0.004). The HS(a) specialist neurons also respond reliably to 1.6 x 10-m M Z 9 14:Ac and 2.0 x 10 ~o M Z 5 - 1 2 : A c alone (Figure 3). These stimulus intensities exceed the level emitted by females, and the response was expected (Mayer, 1993). These two pheromone components also synergized the response to 2.0 × 10 ~3 M Z 7 - 1 2 : A c (Figure 3). The stimulus levels of these two compounds necessary to synergize the response to Z T - 1 2 : A c exceed by two to four orders of magnitude the level emitted by females. Further work is necessary to demonstrate whether or not these compounds might synergize the response at physiological levels. To ascertain both that the synergism observed in the receptor neurons is reflected in behavior and to measure how closely the proportion of sex phero-

T. ni SYNERGISM

1883

mone-elicited behaviors reflect the responses of the HS(a) specialists, flight behaviors and copulatory responses were measured in a wind tunnel at nearly the same stimulus levels that were used in the electrophysiological measures (Table 2). The three concentrations of Z 7 - 1 2 : A c used all elicited submaximal upwind flight behaviors. In support of the etectrophysiological measured synergism, the proportion of males flying upwind and copulating increased with increase in the amount of Z 7 - 1 2 : A c at constant levels of all four synergists. Additionally, the flight response also generally increased proportionally with the increase in amount of the four synergists at constant levels of Z 7 - 1 2 : A c .

DISCUSSION AND CONCLUSIONS The data obtained by both electrophysiological and behavioral experiments are consistent with and support the conclusion that the HS(a) specialist response to Z7-12 :Ac is synergized by two synthetic pheromone analogs and at least two sex pheromone components. There are several facets to specialist synergism that impact such diverse areas as receptor interactions, behavior, and the identification and practical field use of sex pheromones that are significant and merit further comment. Receptor hueractions. The receptor neuron's response begins when pheromone molecule(s) penetrate minute sensillar pores (Slifer et al., 1959: Schneider et al., 1964; Kaissling and Priesner, 1970) and bind to a carrier protein (Riddiford, 1970; Vogt and Riddiford, 1981; Steinbrecht et al., 1992; Laue et al., 1994) and/or inositol-l,4,5-triphosphate-coupled receptors in the dendritic membrane of the receptor neuron (Zufall and Hatt, 1991; Yoshii et al., 1992; Wegener et al., 1993; Stengl, 1994; and Schleicher et at., 1994). The stimulusinduced cascade of molecular events ultimately leads to action potentials that convey information directly to a macroglomerulus in the deutocerebral lobe of the brain (Boeckh et al., 1976; Hildebrand et al., 1980; Christensen et al., 1991). Based on what is now known about receptor interactions, we consider that synergism most likely reflects a simultaneous reaction of Z 7 - 1 2 :Ac and the synergen with a receptor macromolecule. There appears to be a relative lack of specificity required of the synergen's structure in the T. ni receptor because the double bond can be located on a side chain. Nevertheless, there is not a total lack of synergen specificity because some synergens elicit a greater response than others. This is demonstrated by the quantitative differences in responses elicited by mixtures of Z 7 - 1 2 : A c and 6-VD or 10-VT. While both of these synthetic analogs synergized the response, 10-VT elicits about the same response as 6-VD but at about 100 times lower airborne concentration (Table 1). Structurally, the 10-VT molecule has four more methylene groups between the double bond and the acetate group than the

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MAYER AND DOOLII-rLE

10-VINYLTETRADECYL ACETATE

6-VINYLDECYL ACETATE

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Synergism of an insect sex pheromone specialist neuron: Implications for component identification and receptor interactions.

Extracellular recordings show that the response to the sex pheromone component, (Z)-7-dodecenyl acetate (Z7-12:Ac) by the HS(a) antennal olfactory spe...
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