Journal of Chemical Ecology, Vol. 12, No. 2, 1986

INSECT OLFACTORY RECEPTOR RESPONSES TO COMPONENTS OF PHEROMONE BLENDS

ROBERT J. O ' C O N N E L L , J E F F R E Y T. B E A U C H A M P , and A L A N J. G R A N T

The Worcester Foundation for Experimental Biology Shrewsbury, Massachusetts 01545 (Received April 20, 1985; accepted August 1, 1985) Abstract--Multicomponent pheromone systems are common in many insect species. As our knowledge about the number of different chemical compounds actually involved in a particular communicationsystem increases, so too does the need for an efficient neural mechanismfor the encoding of behavioriallyrelevantodor compounds. Here we consider the electrical activity of olfactory receptor neurons in a subset of the individual pheromone-sensitive sensilla on the antennaeof male cabbage looper moths (Trichopluyia hi). Responses to single- and multiple-componentstimuli, drawn from seven behaviorally active compounds, were obtained at several different intensities. Some blends elicited electrical responses which were not readily predicted from a knowledge of the receptor neuron's response to individual components. Key Words--Cabbage looper, Trichoplusia ni, Lepidoptera, Noctuidae, insect attractant, electrophysiology, olfactory receptor, pheromoneblend.

INTRODUCTION The chemical communication system of Trichoplusia ni, the cabbage looper moth (CL), is now k n o w n to involve at least seven behaviorally relevant odor compounds, six of which are produced by the female (Bjostad et al., 1984). As has been the case in a wide range of species, the number of components identified in the female gland of T. ni has increased over the years, keeping pace with technical developments in m o d e m chemistry. In particular, the recent advances in microanalytical procedures have made it possible to monitor the composition of the effluvia from a single behaviorally active female (Baker et al., 1981; Guerin et al., 1981; L6fstedt et al., 1982). This contrasts with the sac451 0098-0331/86/0200-045 I$05.00/0 9 1986 Plenum Publishing Corporation

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rifice of 500,000 virgin females required in Butenandt's original identification of bombykol in the domestic silkworm (Butenandt, 1963). As our awareness of the behavioral and chemical complexity of an insect's pheromonal communication system expands, so too should our appreciation for the demands made upon its nervous system (Card6 and Baker, 1984; Dethier, 1971). For example, it would be reasonable to postulate that the insect olfactory system would be characterized by: extreme specificity at the molecular level, so that exceedingly small changes in the chemical structure of pheromone compounds can be discriminated; a reasonable amount of neural plasticity, so that the molecular diversity that is to be processed can continue to increase as other organisms, including organic chemists, synthesize new behaviorally relevant compounds; enormous gain, so that very dilute, but evolutionarily important, chemical signals, like those emanating from willing but remote sexual partners, can be detected; considerable resolving power, so that these signals, which are likely to be buried in a sea of other nonsignificant compounds, can be discriminated; wide dynamic range, so that small differences in local concentration gradients may be used to locate the sources of distant chemical signals; and, finally, all of these requirements must be packed within the space allocated to the nervous system and fueled within the limited energy budget imposed by the overall metabolic cost of existence. The sensory processing of complex chemical signals by the insect olfactory system has often been postulated to involve a set of narrowly tuned, highly specific olfactory receptor neurons, one for each of the behaviorally relevant component compounds in the pheromone blend (Priesner, 1979a,b, 1980, 1983). In experiments where individual components of a pheromone blend are used as stimuli for single olfactory receptor neurons, many of the behaviorally relevant components do seem to have their own, narrowly tuned specialized receptor neuron type, each apparently dedicated to detecting and signaling its presence (L6fstedt et al., 1982). Even in those instances where several additional compounds may stimulate the same single-receptor neuron, the concentrations required for threshold electrophysiological responses are often so large that a behaving animal would likely never encounter them in sufficient amounts to elicit meaningful neural activity. Thus, each behaviorally relevant component of a complex pheromone blend is thought to have its own private channel of communication with the central nervous system. There the composition of a particular stimulus mixture may be unambiguously decoded by simply noting which of the separate input channels are activated. From a theoretical point of view, this type of system is an efficient neural encoder, only in those cases where the pheromone blend consists of only a few compounds. That is, as the number of compounds in the communication system increases, so too does the requirement for additional private input channels (O'Connell, 1975, 1981). Moreover, in any system where all of the primary receptor neurons are very narrowly tuned, the ability to respond to small amounts

INSECT RESPONSES TO PHEROMONE BLENDS

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of other compounds, including those produced by potential competitors, is severely restricted and can include only those chemicals with nearly identical structural and chemical properties to those which are already in the stimulus set. Thus, a system which relies on highly specialized, narrowly tuned receptors to encode information about pheromones is less adaptive than other arrangements in the sense that it imposes a form of odor blindness on the nervous system which restricts its ability to process the pheromonal signals emanating from other sources. In contrast, efficient encoding of a very large number of different odor compounds can be accomplished, on theoretical grounds, by a relatively small number of different olfactory receptor neuron types if each group is uniquely responsive to a range of different odors (Erickson, 1982; O'Connell, 1981). The identity of a particular stimulus may then be unambiguously decoded by the central nervous system if it evaluates in parallel the relative amounts of neural activity elicited across the whole population of receptor neuron types. It is important to note that the relative breadth of tuning among the different classes of olfactory receptor neurons need not be constant in this type of encoding mechanism. Thus, some fraction of the available input channels could be more narrowly tuned than others. Although highly specific olfactory receptor neurons, apparently tuned to individual behaviorally relevant pheromone components, are observed in many species, it is common to find systems in which behaviorally relevant compounds are processed in the absence of such tuned receptor neurons (Lffstedt et al., 1982; O'Connell, 1972, 1975). There are also insects in which olfactory receptor neurons exist in males that are apparently tuned to compounds that are not produced by the species female (Lrfstedt et al., 1982; O'Connell0 1985c; Priesnet, 1979a,b). Given the chemical complexity of many pheromone systems and the well founded desire to use them as ecologically sound methods of insect pest control, it is absolutely necessary that the neural mechanisms underlying, and responsible for, pheromone perception become the objects of intensive study. One of the basic problems to be evaluated may be stated quite simply: given that the olfactory communication systems of insects are, in nearly all cases, sophisticated (with multiple chemical signals conveying a variety of interrelated messages each of which ultimately results in a series of integrated, context-specific behavioral responses), how does the insect nervous system detect, encode, and process these messages unambiguously and still cope with the range of theoretical restraints enumerated earlier? An initial step toward answering this admittedly global question involves a consideration of the specific response properties of particular classes of insect olfactory receptors when they are each presented with behaviorally relevant components of the pheromone blend. The earliest component identified from female CL (Ignoffo et al., 1963;

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Berger, 1966), and the major volatile component of the gland is (Z)-7-dodecenyl acetate (Z7-12 : Ac). This compound is essential for flight initiation in males and was originally thought to be entirely responsible for the biological activity obtained with female gland extracts. Subsequently, (Z)-7-dodecenol (Z7-12:OH) was shown to significantly arrest upwind flight in males exposed to either calling females or to sources of synthetic Z7-12:Ac (Toba et al., 1970; Tumlinson et al., 1972; McLaughlin et al., 1974). Although this compound has occasionally been found in gland extracts, it is generally considered to arise artificially in the process of chemical separation or analysis (Linnet al., 1984). It is thus classified in common with other behaviorally active compounds which are not components of a female's pheromone blend, as an interspecific compound, perhaps one involved in some speciation mechanism which includes the CL (Fletcher-Howell et al., 1983; Leppla 1983). A saturated 12-carbon acetate, dodecyl acetate (12 : Ac) was next identified in female glands and was Shown to modulate several of the close-range search behaviors of males, including landing frequency and total time spent on the pheromone source (Bjostad et al., 1980). Recently four additional compounds were found in the female gland. They have been identified chemically and implicated behaviorally as important components of the pheromone blend (Bjostad et al., 1984). These compounds include: (Z)-5-dodecenyl acetate (Z5-12 : Ac); 11-dodecenyl acetate (11-12 : Ac); (Z)-7-tetradecenyl acetate (Z7-14 : Ac); and (Z)-9-tetradecenyl acetate (Z9-14:Ac). Although the exact roles of these latter four compounds in modulating normal male behavior is still being evaluated, there is little doubt that, in wind-tunnel assays, the two previously identified CL female components elicit only about 25 % of the close-to-source behaviors observed when animals are exposed to the total complement of the six femaleproduced components (Bjostad et al., 1984; Linnet al., 1984). Moreover the more complex six-component synthetic blend elicits behavioral responses which are quantitatively equivalent to those elicited by excised virgin female pheromone glands, both in terms of the number of males responding and the amount of time required for the full behavioral sequence. The experiments and discussion reported here continue our attempts to unravel blend perception in the peripheral olfactory system of the cabbage looper moth. We had previously focused on the three original compounds whose behavioral import was known (O'Connell, 1985b,c). There we focused especially on the quantitative response characteristics of a selected subset of pheromonereceptor neurons (HS sensilla) which had earlier been shown to be very responsive to Z7-12:Ac and Z7-12:OH (O'Connell et al., 1983). Here we expand these studies in HS sensilla by considering the alterations in neural discharge engendered in these receptor neurons by the addition of the four recently identified pheromone components to the various blends previously considered. The effect of adding the behavioral inhibitor Z7-12 : OH to some of these mixtures

INSECT RESPONSES TO PHEROMONE BLENDS

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was also examined. The number of stimulus combinations was kept manageable by fixing the intensity and composition ratios of the various components at levels near those found in female gland extracts. Since we have explored only a limited portion of the available behaviorally relevant stimulus domain, in only one of the two recognized classes of pheromone-sensitive sensilla, it is not yet possible to provide a thorough description of potential coding mechanisms. However, we hope to show here additional instances where olfactory receptor neurons are responsive, in novel ways, to multiple-component stimuli even in those cases where an individual behaviorally relevant pheromone component is not processed by a separate unique class of receptor neuron.

METHODS AND MATERIALS

Animals. All of the adult moths used in this study were derived from the laboratory-reared stock maintained by the Insect Attractants, Behavior and Basic Biology Research Laboratory, USDA, Gainesville, Florida. Eggs were collected from filter papers placed in laying cages, washed in dilute sodium hypochl0rite solution, and rinsed with distilled water. After hatching, larvae were supplied with ad libitum Velvetbean Caterpillar Diet (#9795, Bio-Serve Inc., Frenchtown, New Jersey 08825). Newly pupated animals were sexed, and experimental males were isolated from further contact with females. Virgin adult males had access to 5 % sucrose solution and were used as subjects when 2-5 days of age. All animals were maintained at 23 ~ 70% relative humidity, and exposed to a 14 : 10-h light-dark cycle. Recordings. Animal preparation, tungsten microelectrode construction, extracellular recording conditions, odor cartridge placement, and their control were as described elsewhere (O'Connell, 1975, 1985c; O'Connell et al., 1983). Briefly, males were restrained and fixed in a recumbent position on a Plexiglas holder. One of their antennae was held in place with transparent adhesive tape and then viewed at 600 power with the long working distance objectives of a light microscope. Transillumination of the antennae revealed the shafts and bases of individual olfactory sensilla and allowed the positioning of electrolytically sharpened recording microelectrodes. In general, we found that stable recordings were routinely obtained with these techniques and that odor-induced responses could be monitored for considerable time periods, apparently limited only by the gradual dehydration of the animal over a 24- to 96-hr interval. The electrical signals generated by the two olfactory receptor neurons found in each sensillum were processed with an AC-coupled amplifier (bandpass 0.33.0 kHz), displayed on an oscilloscope, and then sorted and timed by an online digital computer (O'Connell et al., 1973). For each stimulus application the computer produced: (1) an event-time histogram; (2) a reconstructed spike

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record; and (3) the total number of action potentials elicited in the prestimulus, stimulus, and poststimulus intervals for both receptor neurons in a sensillum. All the data were obtained from sensilla whose morphological and physiological properties classified them as HS sensilla (O'Connell et al., 1983). Stimulation. All the compounds used here are found in the female cabbage looper pheromone gland or are known to modulate male behavior. Both Z 7 12 : Ac and Z7-12 : OH were obtained from Farchan Chemical Co., Columbus, Ohio. The saturated compound, 12:Ac was a gift from Dr. W. Roelofs, New York State Agricultural Experiment Station, Geneva, New York. The purity of these materials was evaluated prior to use by Albany International, Controlled Release Division, Needham Heights, Massachusetts. Based upon gas chromatographic retention times on polar (SP-2340) and nonpolar (SPB-1) columns (60M, WCOT), each calibrated against authentic standards, the level of cross-contamination among these three compounds was negligible (detection limit, < 0.1%). Absolute purity was 87 % for Z7-12 : OH ( + 8 % E7-12 : OH), 93% for Z 7 - 1 2 : A c ( + 5 % E 7 - 1 2 : A c ) , and 98% for 12:Ac. The remaining female produced compounds, l l - 1 2 : A c ; Z5-12:Ac; Z 7 - 1 4 : A c , and Z914:Ac, were a gift from Dr. M.S. Mayer, Insect Attractants, Behavior and Basic Biology Research Laboratory, USDA, Gainesville, Florida. A range of stimulus intensities for each compound was produced by serial dilution with light mineral oil (Aldrich Chemical Co., Milwaukee, Wisconsin). The dilution series varied in decade steps from 0.1 ~g/tzl to 0.0001 t~g/~zl and were known to span the range of physiologically effective intensities typical for HS sensilla. The relative amount of each compound present in a synthetic mixture was matched to those observed analytically in extracts of female glands except for Z7-12 : OH which, although behaviorally active, seems not to be a natural glandular constituent. This latter compound was evaluated in mixtures at a proportion equal to that used with mixtures containing Z7-12:Ac. The various single and multicomponent stimuli were produced by spotting, in turn, 0.5 tzl of the appropriate individual dilution series onto a filter paper (160 mm 2) held in individual glass odor cartridges. Make-up mineral oil, where required to equalize the evaporative surface area, was added to each filter paper so that total liquid volume for each cartridge was 1.5/~1. This method of stimulus preparation results in single cartridges that evoke responses equivalent to those that are obtained when the individual components of a blend are kept in separate cartridges and mixed in the air space surrounding a single sensillum. Thus, within our ability to measure differences between these two modes of stimulation, a single-odor cartridge delivers multiple-component stimuli with individual release rates equivalent to those obtained with cartridges containing only a single pheromone component. Most mixtures were evaluated at a single concentration and composition ratio. The concentration level normally selected was one which would, on average, result in a half-

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INSECT RESPONSES TO PHEROMONE BLENDS

maximal action potential discharge. This allowed us to detect both increases and decreases in the neural activity evoked by mixtures. For the principal component o f the female pheromone blend, Z 7 - 1 2 :Ac, the 0.005 #g/cartridge was routinely used. The six female-produced compounds were made up as a single stock solution at the composition ratios observed in extracts of female glands (Table 1) to minimize the total volume involved when these compounds were evaluated. Completed odor cartridges were sealed with Teflon plugs and caps and stored in individual Teflon-capped glass vials which were, in turn, stored in an opaque container continuously purged with dry nitrogen (1.6 liters/min). Between experiments this container was purged and stored at 4~ These are necessary precautions for pheromone storage and handling and are designed to prevent cross-contamination between individual samples. Individual stimuli were 2 sec in duration and applied, under computer control, by passing purified, oxygen-free nitrogen (60 ml/min) over the filter paper contained in each glass odor cartridge. The outlet of the odor cartridge was positioned 8 m m from the antennal surface. In the interstimulus interval (usually 5 rain), the antenna was bathed by an opposed, humidified pure air stream (Ultra Zero grade; Matheson, Gas Products, East Rutherford, New Jersey), at a flow rate of 120 ml/min. Each stimulus was presented in duplicate to each olfactory receptor neuron. The order o f stimulus presentation proceeded from cartridges containing only single compounds through to those containing all seven compounds. All the responses reported here were obtained with a single set of odor cartridges. Control odor cartridges, which contained only 1.5 #1 of mineral oil on filter paper, were interspersed with odor stimuli on a random basis to monitor for inadvertent contamination of the odor line connections. In the few cases where contamination was revealed by measurable responses to control car-

TABLE l . COMPOSITION OF SIx-CoMPONENT

Compound Z7-12 : Ac Z5-12 : Ac 12 : Ac ! 1-12 : Ac Z7-14 : Ac Z9-14 : Ac

Trichoplusia

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0.00408 0.00037 0.00034 0.00012 0.00006 0.00004

aAverage proportion (relative to Z7-12 : Ac) measured in the gland extracts of six individual females (Bjostad et al., 1984).

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tridges, replacement of the relevant Teflon fittings eliminated subsequent responses to controls. In these cases, all of the response data since the preceding control cartridge presentation were discarded and the individual stimuli were repeated. Thus, there was no need to correct any of the response measures reported here for background responses associated with the mechanics of stimulus application. Data Analysis. The response measure reported here was derived by subtracting the number of action potentials produced by a particular receptor neuron during the 10 sec immediately preceding the 2-sec stimulus pulse from the number of action potentials which occurred in the succeeding 10 sec. The duplicate responses obtained with each odor cartridge were averaged to provide the data set for individual neurons. Because of the large number of possible stimulus combinations among the seven relevant compounds, the range of effective stimulus concentrations likely to be appropriate for each, and the range of potentially interesting composition ratios among the constituents of a particular mixture, the number of individual stimuli evaluated in a single recording session (2 hr) was necessarily only a small fraction of the available stimulus set. In each case, the responses to multicomponent stimuli were compared to the algebraic sum of the responses elicited in the same receptor neurons by the appropriate intensities of their single components. The resulting pairs of expected and observed values were compared statistically with the Witcoxon matched-pairs signed-ranks test or with the sign test (Siegel, 1956). RESULTS

Response Characteristics. Action potentials were recorded extracellularly from sensilla (HS) on the male CL antenna which contains receptor neurons that are highly sensitive to the major pheromone component, Z7-12:Ac. The neurophysiological and morphological characteristics of this class of sensilla have been described and contrasted with the properties of the other class of pheromone-sensitive sensilla on the antenna (O'Connell et al., 1983; Grant and O'Connell, 1985). Each HS sensillum is innervated by two spontaneously active receptor neurons that produce action potentials which can be reliably differentiated from each other by their amplitudes and waveforms. By convention, the receptor neuron producing the larger amplitude action potential is designated the A neuron and that producing the smaller is designated the B neuron. The mean number of spontaneous action potentials (+ SEM) in this sample of 26 individual sensilla was 0.28 + 0.05 impulses/sec for the A receptor neurons and 0.33 + 0.06 impulses/sec for the B receptor neurons. In addition to this typical range of spontaneous activity, individual HS sensilla were also characterized by their length, shape, and responsiveness to low doses (0.0005-0.005 /~g) of Z7-12 : Ac and Z7-12 : OH.

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INSECT RESPONSESTO PHEROMONEBLENDS

Responses to Individual Components. Five of the seven behaviorally relevant compounds have been evaulated as individual stimuli. Their intensities were adjusted to approximate those measured in female pheromone glands (Table 1), except for Z 7 - 1 2 : O H which is not produced by the female and thus was evaluated at an intensity equal to that used for Z7-12 :Ac. As demonstrated in previous studies (Grant and O'Connell, 1985; O'Connelt, 1985a-c; O'Connell et al., 1983) and illustrated here in Figure 1 (taken from O'Connell, 1985c), the average A receptor neuron in HS sensilla is reliably responsive to low doses (0.005 #g) of Z7-12 : Ac whereas the average B receptor neuron is responsive to comparable amounts of Z7-12 :OH. Neither receptor neuron is particularly responsive to 12:Ac, even when larger intensities are evaluated (Figure 1). Similarly, we show in this sample of olfactory receptor neurons that neither Z512:Ac (0.0005 #g) nor Z 7 - 1 4 : A c (0.00005 /zg) alone, elicited consistent amounts of electrical activity in either receptor neuron (Figure 2). Responses to Mixtures. The average magnitude of the electrical response obtained from individual receptor neurons to stimulation with the various mixtures is displayed in Figure 2. The response obtained in each receptor neuron to stimulation with a particular mixture was compared to the response expected if the responses to individual components were additive. The observed and expected response values for each receptor neuron were then compared to each other with the Wilcoxon matched-pairs signed-ranks test. In general, the direction of the statistically significant trends observed in the responses elicited by

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mixtures was similar in both the average A and B receptor neuron. Of the five binary mixtures evaluated in this sample of HS sensilla, three elicited responses which were significantly different from the expectations of the additive model (starred bars in Figure 2). We previously demonstrated in another sample of 47 sensilla that 12 : Ac has a significant synergistic effect on the magnitude of the responses elicited by Z 7 - 1 2 : A c in both the A and B receptor neuron of the average HS sensillum (O'Connell, 1985b,c). We show in this sample of sensilla, similar levels of synergy and note that 12 : Ac seems unique in this regard because the other two minor pheromone components examined (Z5-12 : A c and Z 7 - 1 4 : A c ) did not significantly alter the responses elicited in either receptor neuron when they were individually combined with Z 7 - 1 2 : A c . The responses elicited in both receptor neurons, by a blend containing all six of the identified female-produced compounds, were, on average, indistinguishable from those elicited by the binary mixture containing only Z 7 - 1 2 : Ac and 12 : Ac. In addition, the average magnitude of the A receptor neuron discharge elicited by this blend is significantly larger than would be expected from the additive model.

INSECT RESPONSES TO PHEROMONE BLENDS

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The responses elicited by mixtures which contain Z7-12 : OH are, on average, reliably smaller than those predicted by summing the responses obtained with the individual components of the mixture. The three-component blend (I + II + III, see Figure 2 legend) elicited responses which were, on average, smaller than those obtained with the most effective single component. The one exception observed here involved the mixture containing all seven behaviorally active compounds. In this case both receptor neurons produced responses to this complex blend which were, on average, equal to those expected from the additive model (Figure 2). The alterations in average discharge magnitude evoked in the receptor neurons by addition of Z 7 - 1 2 : O H to stimuli containing various components of the pheromone blend were examined in more detail by computing a series of average response histograms. These histograms were obtained by averaging the response histograms obtained from 12 individual sensilla, each stimulated with the indicated compounds. They display the average number of impulses which occurred in successive 100-msec bins for the 5 sec immediately following stimulus onset and provide a convenient summary of the average pattern of discharge evoked by a particular stimulus. On the left of Figure 3 we show the average responses evoked by several single and multicomponent stimuli in this sample of twelve A receptor neurons. On the right are the responses obtained in the same set of neurons for these mixtures, now with the inclusion of 0.005 #g of Z7-12 : OH. Although these response histograms are averages of the activity elicited in these 12 neurons by the indicated components, it is clear that they retain characteristic differences in both the amount of neural activity evoked and its temporal distribution from one stimulus to another. In part, these observations are in agreement with those of Grant and his colleagues who showed that individual receptor cells tend to have unique temporal patterns of discharge. Some receptor neurons respond to stimulation with a fixed dose of a selected pheromone component by producing a reasonably steady tonic level of discharge which may persist for the duration of the stimulus exposure. Others will respond to the same stimulus with an initial phasic burst of activity followed by a more prolonged tonic level of discharge even in situations where it is known that rectangular pulses of pheromone are being provided (Grant et al., 1985). The histograms of Figure 3 also reinforce our earlier observation that the addition of 10% 12:Ac to Z 7 - 1 2 : A c (Figure 3B) greatly enhances the amount of neural activity elicited in A receptor neurons especially when compared to the amount of activity ascribable to the presence of Z7-12 : Ac alone (Figure 3A). This increase is even more impressive in light of the relatively narrow tuning of A receptor neurons previously demonstrated in this species and the general failure of 12 : Ac to be an effective single stimulus for the two receptor neurons in HS sensilla (O'Connell et al., 1983). This ability to synergize the responses elicited in A receptor neurons is unique to 12 : Ac since the addition

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of the remaining four female-produced compounds to the blend (Figure 3C) did not elicit an additional increase in the amount of discharge obtained when compared to that evoked by stimulation with the binary mixture (Figure 3B). However, these additional materials are not without impact on neural activity because they clearly prevent the significant reduction in discharge expected when Z7-12 : OH is added to the total blend (Figure 3F). That is, the addition of Z T 12:OH to the binary mixture containing Z7-12 : Ac and 12 : Ac normally results in a net reduction in the response evoked by the trinary mixture (Figure 3B and E). However, the response elicited by stimulation with the total blend plus Z 7 12:OH (Figure 3F) remains equivalent to that obtained with the total blend alone (Figure 3C). The effects of these alterations can be more easily appreciated by subtract-

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ing the appropriate average response histograms from each other to produce difference histograms as shown in Figure 4. Again we see that there are two significant interactions, one involving the increase elicited by the addition of 1 2 : A c to Z 7 - 1 2 : A c (Figure 4B) and a second involving the large reduction elicited by the addition of Z 7 - 1 2 : O H to this binary mixture (Figure 4E). In both cases the total amount of neural discharge and its temporal pattern have been substantially altered. There is also a tendency for the alterations observed to be larger in the earlier portions of the neural discharge. It is also clear that the newly identified female-produced pheromone components do not add substantially to the response obtained with the binary mixture (Figure 4C), yet they do prevent the reduction in response expected subsequent to the addition of Z 7 12 : OH to the female blend (Figure 4F).

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SECONDS FIG. 4. Difference histograms which plot in 100-msec bins the number of action potentials remaining after two average response histograms (shown in Figure 3) have been subtracted from each other. Each of the bins with statistically significant difference scores (P < 0.05, one-tailed, sign test) are indicated in black. The response histograms subtracted are: (A) Z 7 - 1 2 : A c + Z7-12:OH - (Z7-12:Ac + Z7-12:OH + 12:Ac); (B) Z7-12:Ac + 12:Ac - (Z7-12:Ac); (C) Z 7 - 1 2 : A c + 12:Ac - (blend); (D) Z712:Ac - (Z7-12:Ac + Z7-12:OH); (E) Z7-12:Ac + 12:Ac - (Z7-12:Ac + 12:Ac + Z7-12:OH); (F)blend - (blend + Z7-12:OH).

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Given the high level of specificity encountered historically in individual insect pheromone-receptor neuron studies, most researchers seem to assume that an N component pheromone blend is processed by N different specialized receptor neurons. Although the interactive aspects of blend perception have not often been considered in detail, multicomponent stimuli, when they have been used, usually fail to evoke unique responses in single receptor neurons beyond those which can be accounted for from a knowledge of their responses to individual components of a blend. A potential contributing factor to this failure to observe interactions among multicomponent stimuli may be the natural tendency to examine only those single-pheromone compounds which are clearly responsible for a well-defined component of male sexual behavior, are known to produce a large summated neural discharge (EAG), or are processed by specialized olfactory receptor neurons. Nearly every species so far examined produces a large number of pheromone-like compounds whose collective behavioral and electrophysiological properties are not explored in the context of a multicomponent pheromone blend because they individually fail to elicit specific behavioral responses or discriminable units of electrical activity from peripheral olfactory receptor neurons. The four pheromone components, recently described in the female cabbage looper gland, provide a good object lesson which demonstrates the rapid increases in the chemical, behavioral, and electrophysiological complexity of pheromone communication systems. As individual stimuli, these compounds fail to elicit any of the normal components of male sexual behavior (Linnet al., 1984). They are also completely inactive when mixed together at intensities comparable to their abundance in extracts of the female pheromone gland. However, addition of the originally identified pheromone component Z 7 - 1 2 : A c to these compounds results in a five-part blend which is fully competitive with calling virgin females. Addition of the sixth female-produced compound 12 :Ac, does not elicit further significant increases in the amount of male behavior observed. If individual single components are removed from the total blend, only Z7-12 : Ac can be associated with a specific component of male behavior. As the complexity of the blend is further reduced, decrements in male behavior begin to occur in a graded fashion. The close-to-source behaviors are more affected than are those further downwind. However, it is still not possible to ascribe the absence or reduction of a particular behavioral sequence with the removal of a specific compound (Linnet al., 1984). It is possible that the behavioral results obtained by Linn and his colleagues (1984) in the CL may represent an isolated case in which the pheromone communication system is unusually complex. However, the ever-growing list of compounds identified in individual female pheromone glands suggests that their

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results m a y be the n o r m (Baker and Card6, 1979; B a k e r et al., 1976; Bjostad and R o e l o f s , 1983). A l t h o u g h w e h a v e only b e g u n to e v a l u a t e the p h y s i o l o g i c a l responses elicited in o n e o f the two subsets o f p h e r o m o n e - s e n s i t i v e sensilla by stimulation with o n l y a few o f the possible blends a m o n g the s e v e n b e h a v i o r a l l y active c o m p o u n d s , six o f w h i c h are f e m a l e - p r o d u c e d p h e r o m o n e c o m p o n e n t s , it is clear that o u r c o n c e p t u a l i z a t i o n o f the neural basis for p h e r o m o n e d i s c r i m ination m u s t be greatly e x p a n d e d . M i x t u r e interactions o f the sort described here, at the l e v e l o f p r i m a r y olfactory r e c e p t o r neurons, p r o v i d e s appropriate grist for a s y s t e m in w h i c h m o r e c o m p l e x c o d i n g m e c h a n i s m s m a y occur. Acknowledgments--These studies have been supported by grants NS 14453 from NINCDS, BNS 8016395 from NSF, and by the Alden Trust. We thank S. Treistman for comments on the manuscript, M. Eaton for her assistance, and M.S. Mayer for periodic infusions of pupae and pheromone components.

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