Pheromone Biochemistry

reality.

I

PHEROMONE BIOSYNTHESIS AND ITS REGULATION

Outline

Chapter 1: Relationship of Structure and Function to Biochemistry in Insect Pheromone Systems

Chapter 2: Biology and Ultrastructure of Sex Pheromone–Producing Glands

Chapter 3: Pheromone Biosynthesis in Lepidopterans: Desaturation and Chain Shortening

Chapter 4: Pheromone Biosynthesis: Enzymatic Studies in Lepidoptera

Chapter 5: Endocrine Regulation of Pheromone Production in Lepidoptera

Chapter 6: Biosynthesis of Pheromones and Endocrine Regulation of Pheromone Production in Coleoptera

Chapter 7: Biosynthesis and Endocrine Regulation of Sex Pheromone Production in Diptera

Chapter 8: Alkaloid-Derived Pheromones and Sexual Selection in Lepidoptera

Chapter 9: Neuroendocrine Regulation of Sex Pheromone–Mediated Behavior in Ixodid Ticks

Chapter 10: Cantharidin Biosynthesis and Function in Meloid Beetles

1

Relationship of Structure and Function to Biochemistry in Insect Pheromone Systems

J.H. TUMLINSON and P.E.A. TEAL,     Insect Attractants, Behavior, and Basic Biology Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, Florida 32604

Publisher Summary

This chapter discusses the relationship of structure and function to biochemistry in insect pheromone systems. It presents molecular studies in pheromone biosynthesis, perception, and catabolism. A wide range of compounds have been identified as insect pheromone components. While carbon, hydrogen, and oxygen are the usual atoms incorporated into these molecules, nitrogenated and chlorinated compounds have also been identified. Usually, small molecules are used for communication when rapid dispersal of the signal is needed, while larger, less volatile compounds tend to function in attraction and stimulation when prolonged exposure is necessary. The former case is exemplified by 4-methyl-3-heptanone, used by numerous species of myrmicine ants as an alarm pheromone. The latter is illustrated by numerous lepidopteran sex pheromone components that are generally between 10 and 24 carbons in length and are much less volatile than the alarm pheromone. In addition to the different molecular sizes that reflect behavioral functions, pheromone structures vary greatly between different orders of insects. Generic themes of structural type exist within groups as is evidenced by the use of the same or structurally related compounds by many species of the same genus. These themes are the result of the development of common biosynthetic pathways with differences in blend ratios and components being the result of minor permutations in the enzymatic steps involved.

I INTRODUCTION

Chemical cues are major sources of information used by most insects to interpret environmental stimuli. This reliance on chemical stimuli undoubtedly stems from the development of chemosensory organs and cells early in evolutionary history, perhaps even before the development of light-sensitive organs (Snodgrass, 1926). Broadly speaking, these chemical stimuli are categorized as semiochemicals. They function as pheromones when used for intraspecific communication. When used at the interspecific level, they are termed kairomones when the species responding to the chemical message benefits and allomones when the species emitting the signal gains some advantage over the receiving organism. There is considerable overlap between these classes, and often the same compounds serve both intra- and interspecific functions. Therefore, in order to elucidate the roles of individual semiochemicals it is necessary to study all aspects of the communication system from biosynthesis of the compounds to the perception and integration of the compounds by all of the organisms responding.

Of the three classes of semiochemicals mentioned above, pheromones are the most extensively studied. Although all insect orders use pheromones in communication, the highly social Hymenoptera and Isoptera have developed the most complex and sophisticated pheromone systems. In fact, Blum (1974) suggests a strong evolutionary relationship between the development of insect societies and diversification of pheromone communication. Among subsocial insects, pheromones have been shown to play major roles in (1) the initiation of gregarious behavior during group oviposition among certain mosquitoes (Hudson and Mclintock, 1967) and the desert locust Schistocerca gregaria (Forsk.) (Norris, 1963); (2) the formation of aggregations at food sites, particularly among scolytid beetles (Birch, 1984) and Drosophila species (Bartelt et al., 1985); (3) dispersal behavior among generally gregarious species during predator attack (Nault and Phelan, 1984); (4) the synchronization of gamete maturity among species exhibiting aggregative behaviors (Blum, 1974); and (5) mate attraction among species that maintain a solitary life style.

According to Inscoe (1977), conspecific attractancy among lepidopteran species has been known since 1690, when John Ray reported several male Biston betularia (L.) flying around a caged female. This knowledge of the attractive capacity of female Lepidoptera also was used by such great naturalists as Fabré for collection of rare specimens; the procedure used was essentially the same as that of Ray (Kettlewell, 1946; Inscoe, 1977). The use of live females for population monitoring of the gypsy moth, Lymantria dispar (L.), began in 1914, but by 1920 the females had been replaced by crude abdominal tip extracts that remained active for longer periods than females (Collins and Potts, 1932). Attempts to isolate the chemical components of lepidopteran sex attractants also began in the 1920s. Unfortunately, the methods then available for chemical analysis were not adequate, requiring large sample quantities and necessitating continuous rearing of large numbers of insects. As a consequence, little headway was made. The first sex pheromone identified was that of Bombyx mori (L.), the silkworm moth, by Butenandt et al. (1959). The elucidation of bombykol [(E,Z)-10,12-hexadecadien-1-ol] required 20 years and 500,000 female abdomens.

Subsequent to the identification of bombykol, considerable emphasis was placed on the identification of pheromone components of pest Lepidoptera, and, in 1966, (Z)-7-dodecenyl acetate was identified as the sex pheromone of the cabbage looper moth (Berger, 1966). Following this, single components of the pheromones of a number of noctuid and tortricid moths were identified. This led to the magic bullet theory of pheromone communication, which hypothesized that every insect species used a single compound for pheromone communication and that each species was isolated from closely related species by differences in the functionality or number and geometry of double bonds within the pheromone molecule. This hypothesis was generally accepted by many researchers working on Lepidoptera until about 1970. However, early work on bark beetles by Silverstein et al. (1966) in which three terpenes, (S)-(–)-ipsenol (I), (S)-(+)-ipsdienol (II), and (S)-(+)-cis-verbenol (III), were identified as a synergistic pheromone blend for Ips paraconfusus Lanier, indicated that multicomponent pheromones were used by Coleoptera. This has since been shown to be true for most insects, and now single-component pheromones are the exception rather than the rule.

Our knowledge of the chemistry, behavior, physiology, and biochemistry of insect communication systems has increased dramatically over the past 25 years. Early studies were aimed at two different goals. The first was development of a basic knowledge about the biological aspects of pheromone communication, as is indicated by the early work of Shorey and co-workers (e.g., Shorey, 1964; Shorey and Gaston, 1965). The second area was the identification and synthesis of pheromones based on simple bioassays. The bioassays used for these studies tended to rely on single behaviors, such as flight or clasper extension, of groups of insects and failed to monitor observations of the whole range of reproductive behaviors exhibited by individual males. Additionally, the chromatographic and spectroscopic instrumentation available in the 1960s was incapable of resolving complex isomeric mixtures or of detecting minor components present in only nanogram amounts. Thus, usually only the components present in greatest quantity were identified. This is illustrated by the identification of (Z)-7-dodecenyl acetate as the sex pheromone of the cabbage looper moth (Berger, 1966). While this compound is an effective attractant for males for this species, the insects do not exhibit the entire range of behaviors performed in response to females. It was not until 1984 that Bjostad et al. (1984) and Linn et al. (1984) accurately defined the complete pheromone blend of this insect. The additional components identified by Bjostad et al. (1984) are present in very small amounts and were not found until studies on biosynthesis identified the precursors of the additional components. This demonstrates the need for studies on all aspects of semiochemical-mediated biology.

The next step in the evolution of studies on pheromone communication came with the development and common use of electrophysiological techniques including the electroantennogram and single cell recording. These studies aided the identification process greatly and, in conjunction with electron microscopic studies, also formed the foundations on which our theories of pheromone perception are based. These advances coupled with tremendous improvements in analytical instrumentation (Heath and Tumlinson, 1984) and the use of flight tunnel studies (see Fig. 1, Section III) to sequentially analyze the responses of individual insects have led us to the realization that insects use complex chemical systems, rather than single unrelated signals, for communication. Frequently there is considerable overlap in signals, particularly among closely related species.

Fig. 1 Typical responses of male noctuid moths to the sex pheromone released by female moths.

While present methods of study have been highly effective in defining the actual pheromone blends released and the behavioral roles of the chemical components, as exemplified by the work of Baker and Cardé (1979) on the oriental fruit moth, Linn et al. (1984) on the cabbage looper, and Teal et al. (1986) on the tobacco budworm moth, we have not been able as yet to completely unveil the total communication system from biosynthesis to perception and neural integration. Although initial studies on biosynthesis of pheromones were begun in the 1970s when Kasang et al. (1974) induced female gypsy moths to synthesize tritiated disparlure and Jones and Berger (1978) succeeded in inducing female cabbage loopers to produce ¹⁴C-labeled (Z)-7-dodecenyl acetate by injecting [1-¹⁴C]acetate, only limited work on pheromone anabolism and catabolism had been conducted until recently. This type of research is needed to provide the insight into the regulation of pheromone production and perception that will allow us to exploit the inherent weaknesses in the communication systems for development of more effective control strategies. The chapters that follow will present stimulating new results of molecular studies in pheromone biosynthesis, perception, and catabolism.

II CHEMICAL STRUCTURE OF INSECT PHEROMONE SYSTEMS

A wide range of compounds have been identified as insect pheromone components. While carbon, hydrogen, and oxygen are the usual atoms incorporated into these molecules, nitrogenated and chlorinated compounds have been identified also (see Tamaki, 1985). Usually, small molecules are used for communication when rapid dispersal of the signal is needed, while larger, less volatile compounds tend to function in attraction and stimulation when prolonged exposure is necessary. The former case is exemplified by 4-methyl-3-heptanone (IV), used by numerous species of myrmicine ants as an alarm pheromone (Moser et al., 1968; Riley et al., 1974). The latter is illustrated by numerous lepidopteran sex pheromone components which are generally between 10 and 24 carbons in length [for example, the lesser peachtree borer, Synanthedon pictipes (Grote and Robinson), pheromone, (E,Z)-3,13-octadecadien-1-ol acetate (V)] and are much less volatile than the alarm pheromone mentioned above.

In addition to the different molecular sizes, which reflect behavioral functions, pheromone structures vary greatly between different orders of insects, and even within orders tremendous diversity in molecular structure exists. Coleopteran species provide an excellent example of this, with structures varying from the terpenes used by bark beetles (Silverstein et al., 1966) and the boll weevil (Tumlinson et al., 1969) to the 8-methyl-2-decanol propanoates used by Diabrotica species (Guss et al., 1982) and the lactone, (Z)-5-(1-decenyl)dihydro-2(3H)-furanone (VI), of the Japanese beetle (Tumlinson et al., 1977). Nonetheless, generic themes of structural type exist within groups as is evidenced by the use of the same or structurally related compounds by many species of the same genus. These themes are the result of the development of common biosynthetic pathways with differences in blend ratios and components being the result of minor permutations in the enzymatic steps involved.

The activity of a semiochemical is imparted by many factors including size, shape, chirality, degree of unsaturation, and the functional group of the molecule. Among many insects the position, number, and geometry of double bonds are critical for activity. Even so, structurally similar molecules may be active although at greatly reduced levels, and positional isomers often have electrophysiological activity (Priesner, 1968). For example, hexalure [(Z)-7-hexadecenyl acetate] is attractive to pink bollworm males but is considerably less active than the 1:1 blend of (Z,Z)- and (Z,E)-7,11-hexadecadienyl acetates, which comprise the pheromone blend (Hummel et al., 1973).

Similarly, the functional group may be changed in some instances with the resulting compounds eliciting behavioral responses similar to those triggered by the actual pheromone components. Formates, for example, have been substituted for the aldehydic pheromone components of some noctuid moths (Beevor et al., 1977; Mitchell et al., 1975, 1978) with apparently little effect on the resulting attraction of males. In these cases, there is some specificity, however, and not all formate analogs mimic the corresponding aldehydes. Thus, (Z)-9-tetradecenyl formate (VII) substitutes for (Z)-11-hexadecenal (VIII) in Heliothis zea (Boddie) and H. virescens (F.) blends. However, when (Z)-7-dodecenyl formate was substituted for (Z)-9-tetradecenal in the H. virescens pheromone blend, H. virescens males were not attracted (Mitchell et al., 1978).

Finally, some molecules differing in both the number of olefinic bonds and the functional moiety can substitute for actual pheromone components. This is demonstrated by the diolefinic hydrocarbon analog, (Z)-1,12-heptadecadiene (IX), of the major aldehyde component (VIII) of the corn earworm (Carlson and McLaughlin, 1982), which is active behaviorally even though it lacks the aldehyde functionality. However, IX had no effect on H. virescens behavior despite the fact that VIII is the major component of both H. virescens and H. zea pheromone blends. Similarly, Silk et al. (1985) showed that (E)- and (Z)-1,12-pentadecadiene, analogs of both (E)- and (Z)-11-tetradecenal, the pheromone of the spruce budworm moth, were active in behavioral experiments.

In all of the above cases the analogs have chemical structures quite similar to the pheromone components. These features show that insect communication systems are not as rigid as once thought, and it has been suggested that this fluidity might be exploited by developing active pheromone analogs that are more economical to synthesize and/or more stable, and that would therefore be of more practical value in pest management (Carlson and McLaughlin, 1982).

While there is a degree of structural flexibility in the molecules that can be perceived by some species, particularly when the molecules are fatty acid-like, the existence of chirality in a pheromone usually imparts a much higher degree of rigidity to the system. Chirality plays a very important role in the specificity of many pheromone molecules, and often one enantiomer gives a positive biological response while the other enantiomer inhibits the behavior. For example, as little as 1% of the S-(+) enantiomer of VI significantly reduces the response of male Japanese beetles to their pheromone, (R)-(–)-VI. The greatest number of chiral pheromones have been discovered in coleopteran species, and examples within this order serve to exemplify a number of the nine possible categories of behavioral response to enantiomers or diastereomers hypothesized by Silverstein (1979). The western corn rootworm provides an example of two of the response categories. The naturally produced pheromone of this species is (R,R)-8-methyl-2-decyl propanoate (X), and males are neither inhibited nor attracted to the 2R,8S or 2S,8S isomers. However, the 2S,8R isomer is attractive, although at a much lower level than the natural 2R,8R (Guss et al., 1984). A closely related species, the northern corn rootworm, is strongly inhibited by the 2S,8R stereoisomer but uses the 2R,8R for communication (Guss et al., 1985; Dobson and Teal, 1987). Thus, when an enantiomer or a single stereoisomer is used for communication, the other enantiomer or stereoisomers may elicit positive or inhibitory responses or may not elicit any response.

Often the communication systems and the role of chirality in these systems can be even more complicated. The pheromone systems of the various strains of Ips pini (Say) serve as a good example. Initially, the pheromone of I. pini in California was identified as (R)-(–)-ipsdienol [the R-(–) enantiomer of II]. As little as 3% of the S-(+) enantiomer, a component of the pheromone of the competitive I. paraconfusus, completely interrupted the I. pini response to the R-(–) (Birch et al., 1980a). However, I. pini in New York produces a 65 : 35 ratio of (+) : (–) ipsdienol and responds more strongly in the field to synthetic racemic ipsdienol (Lanier et al., 1980). A survey of the literature on Ips pheromones indicates that the species in this genus use varying combinations of components and chiralities to achieve the necessary pheromone specificity. The stereospecificity of pheromone biosynthesis in this beetle is summarized by Vanderwel and Oehlschlager (Chapter 6, this volume).

Blends and the regulation of blends also are critical factors in determining pheromone specificity and are, therefore, two of the most important aspects of pheromone biosynthesis. As indicated earlier, the first multicomponent pheromone identified was that used by Ips paraconfusus, composed of (S)-(–)-ipsenol (I), (S)-(+)-ipsdienol (II), and (S)-(–)-cis-verbenol (III) (Silverstein et al., 1966). These compounds were found to be synergistic in that little or no attraction occurred when the individual components were tested, but the combination of the three was highly attractive. Similar cases have been documented for many species, particularly among the Lepidoptera. For example, both (Z)-9-tetradecenal and (Z)-11-hexadecenal act in concert to induce upwind flight by males of H. virescens, but neither component shows significant activity when released alone (Vetter and Baker, 1983). Another interesting feature of pheromone blends is that in many instances the individual components function in concert to maximize each sequential step in the behavioral sequence. Thus, rather than each component being responsible for the elicitation of a single behavior, the essence of the whole blend is required for maximizing the behavioral sequence. This is exemplified by studies conducted by Baker and Cardé (1979) on the oriental fruit moth, Linn et al., (1984) on the cabbage looper, and Teal et al. (1986) on the tobacco budworm moth.

Within closely related taxa variations in blends, or ratios of components in blends, play critical roles in affecting species specificity. Interestingly, differences in these blends reflect very minor differences in the basic biosynthetic processes that produce the components but have major ramifications with regard to the behaviors elicited by the species involved. Mechanisms by which multicomponent pheromones can increase reproductive isolation are many and include (1) the release of a component having no effect on conspecifics but an inhibitory effect on males of another species, (2) the addition of components necessary for reproductive behaviors among conspecific males and which are inhibitory to males of other species, and (3) differences in the component ratios among species using the same chemicals.

Although cases are few in which a component is produced solely for the purpose of decreasing the reproductive responses of males of other species, such a case is indicated between the gypsy moth, Lymantria dispar, and the nun moth, L. monacha (L.). The sex pheromone of the gypsy moth was identified as (Z)-7,8-epoxy-2-methyloctadecane (disparlure, XI) by Bierl et al. (1970). Studies using the (+) and (–) enantiomers and the racemic mixture have indicated that males respond only to the (+) enantiomer and are inhibited by very small amounts of (–)-disparlure (Cardé et al., 1977b; Miller et al., 1977; Miller and Roelofs, 1978). Male gypsy moths have two antennal neurons that respond to the pheromone. However, one neuron responds solely to the (+) enantiomer, while the other cell responds only to (–)-disparlure (Hansen, 1984). The nun moth also is attracted to (+)-disparlure, but studies have indicated that the (–) enantiomer has no effect on the attraction of this moth (Klimetzek et al., 1976; Vite et al., 1976). However, Hansen (1984) reported that extracts of nun moth pheromone glands elicited responses from both receptor cells in gypsy moth male antenna and produced evidence to indicate that the nun moth females produce a 90: 10 blend of (–)- and (+)-disparlure. Therefore, reproductive isolation between gypsy moth males and nun moth females appears to result from the release by the latter species of a compound that is not perceived by conspecific males.

The addition of a compound or compounds required for upwind anemotaxis and/or courtship by conspecific males, and which are inhibitory to males of other species, is particularly evident among the tortricids. For example, the two closely related, co-occurring species, Argyrotaenia velutinana (Walker) and Choristoneura rosaceana (Harris), both use a 9 : 1 ratio of (Z)- to (E)-11-tetradecenyl acetate in their pheromone blends, but in addition to these A. velutinana also produces dodecyl acetate (Cardé et al., 1977a). The addition of the dodecyl acetate increases conspecific male atraction and effectively inhibits the response of male C. rosaceana (Roelofs and Cardé, 1974). Similarly, in the communications systems of I. pini and I. paraconfusus, which are competing species, the pheromone of each functions as an allomone for the other by interrupting response to conspecific semiochemicals (Birch et al., 1977, 1980a,b; Light and Birch, 1979).

The function of differences in pheromone blend ratios in reproductive isolation is also evident among the Tortricidae. Cardé et al. (1977a) have shown it to effect the isolation of two species, Archips argyrospilus (Walker) and A. mortuanus Kearfott, which share four components. Unfortunately, little work has been done on the roles of such ratios, on individual components therein, or on the behavioral steps leading to mating, so that it is unknown if each component performs the same role in each species.

As we have indicated, there is much interspecific communication with pheromones, particularly among closely related species. There is another type of interspecific communication in which a pheromone may function in another capacity. For example, components of the sex pheromone of H. zea also act as kairomones that increase searching efficiency of the egg parasite Trichogramma pretiosum (Riley) (Lewis et al., 1982). Similarly, parasites, for example, Tomicobia tibialis Ashmead (Rice, 1969), and predators, including Temnochila chlorodia (Mannerheim) (Bedard et al., 1969, 1980), both respond to pheromones released by their beetle hosts, I. paraconfusus and Dendroctonus brevicomis LeConte, respectively.

Another complicating factor, largely unexplored, is the interaction between pheromones and host-produced kairomones in chemical communication systems. For example, the aggregation pheromone of the bark beetle, D. brevicomis, consists of frontalin produced by males and exo-brevicomin released by females. These two components, however, are synergized by myrcene released by the tree under attack (Birch, 1984). Another synergistic kairomone–pheromone system has been documented for aggregations of Japanese beetles (Klein et al., 1973, 1980). In this case, the sex pheromone produced by females is synergized by eugenol and phenylethyl propionate, which are found in several host plants of the Japanese beetles. Both males and females are attracted to the combined lure in much greater numbers than to either the pheromone or the plant attractant alone. These cases exemplify complexities inherent to semiochemical communication systems and serve to illustrate the major importance of chemical stimuli for the insect.

III BEHAVIORAL AND PHYSIOLOGICAL RESPONSES OF INSECTS TO PHEROMONES

The bioassay is critical in determining the identity of compounds involved in semiochemical communication. More often than not, the design of the bioassay effectively determines the compounds that will be identified as pheromone components. For example, the use of activation bioassays like that described by Gaston and Shorey (1964) has often led to the identification of only the major components in a pheromone blend, which may have only limited or no activity in the field.

The many difficulties encountered in the development of effective bioassays all relate to the basic semiochemical-mediated behavior of the species in question. Therefore, it is crucial that the whole behavioral sequence involved in communication between the insects be critically evaluated prior to analysis of the behavioral effects of the semiochemicals alone. This behavioral analysis is extremely complicated and is often hampered by variation in environmental parameters such as temperature and humidity. Thus, analysis of semiochemically mediated behavior is often best accomplished in laboratory flight tunnel studies (Fig. 1) (Baker and Linn, 1984). Such assays allow for variations in single parameters such as blend ratios, concentrations, and wind speed while maintaining other factors constant. Thus, single effects can be analyzed, and pheromone components and the roles played by the components in chemically mediated behavior can be identified. The final test, however, is observation under field conditions because of the complex environmental factors that must interact with and modify the effect of the blend in the natural environment (Cardé and Elkington, 1984).

Electrophysiological studies are of considerable value in determining the ability of an insect to perceive semiochemicals. The electroantennogram (EAG) is widely used in isolation and identification studies because of the ease of operation and because a large number of candidate compounds can be screened with this method (Roelofs, 1984). This technique has even been interfaced to capillary gas chromatographs for use as a highly sensitive detector (Struble and Arn, 1984). The EAG indicates only that a compound stimulates receptor cells, however, and does not give information regarding the behavioral response to the compound. Therefore, compounds such as (Z)-11-hexadecen-1-ol, which was identified in pheromone gland extracts of the corn earworm, give significant EAG recordings, but the compound is a potent inhibitor to male behavior when used in pheromone traps (Teal et al., 1984). Furthermore, behaviorally inactive compounds like positional or geometrical isomers often give significant EAG responses (Roelofs, 1984).

Single-cell electrophysiological recordings are much more selective in their responses. These studies involve monitoring responses of single receptor cells within the antennae and, more recently, the responses of neurons within the antennal lobe of the brain. A number of studies have suggested that the sensillar cells that respond to pheromone components, the so-called specialists, have binding macromolecules specific for pheromone molecules (Kaissling, 1979; Priesner, 1979; Mustaparta, 1979; Mayer and Mankin, 1985). Evidence presented by Mayer and Mankin (1985) on the cabbage looper moth suggests that an across-fiber response pattern of the different types of receptor cells within the antenna which respond to pheromone molecules sets up the coding necessary to elicit a behavioral response. Studies also have demonstrated that the responses to two components of a pheromone can be synergistic when the compounds are mixed together (O’Connell, 1972, O’Connell et al., 1986). Therefore, it appears that the first step in pheromonal discrimination occurs in the peripheral nervous system. Studies like that reported by the Matsumoto and Hildebrand (1981) on the tobacco hornworm, Manduca sexta (L.), in which pheromone-responsive neurons in the brain cells were stained, have shown that pheromone-specific neurons are present only in the brains of the responding sex. Therefore, although the behavioral response by females to an oviposition site and males to a sexually receptive female may be identical (Schneiderman et al., 1986), only males respond to the sex pheromone because of the presence of sex-specific receptors in the antenna and neurons in the brain. Studies of these types are critical for developing an understanding of the mechanisms of perception and central nervous system processing of pheromone-mediated events.

Recently, several groups have developed novel methods for probing the mechanisms involved in physiological and behavioral response to pheromones. Schneiderman et al. (1982) successfully grafted the antennal imaginal discs of fifth-instar male tobacco hornworm moths into female larvae and showed that a macroglomerular complex normally found only in the antennal lobes in the brain of an adult male developed in these gynandromorphic females during metamorphic adult development. Furthermore, neurons in these macroglomerular complexes responded when pheromone-sensitive neurons in the transplanted antenna were stimulated with the female sex pheromone. Subsequent studies have indicated that the gynandromorphic females responded to the female-produced sex pheromone in a flight tunnel with the same behavioral pattern shown by normal males (Schneiderman et al., 1986). Studies of this nature have demonstrated sex-specific pathways in the central nervous system which are important in understanding the male responses of insects to pheromones.

Another technique used to probe the mechanism of response to pheromones is the analysis of an insect’s behavior after it is treated with sublethal amounts of neurotoxins. Studies with male oriental fruit moths have established that topical application of sublethal amounts of a number of neuroactive compounds results in changes in specific behaviors associated with male response to pheromones and that a dose–response curve can be established for each neuroactive compound (Linn and Roelofs, 1984). For example, exposure to permethrin caused a significant reduction in the number of males that entered taxis, while chlordimeform affected all of the response categories with hairpencil display being reduced to the greatest extent. Linn and Roelofs (1984) also studied the effect of dual treatment with antagonistic compounds like octopamine and yohimbine and found that normal response was restored in these cases. These studies have demonstrated that the neurotoxins do not affect peripheral reception but rather seem to affect perception by the central nervous system and the motor responses regulated by the central nervous system.

Studies in one of our laboratories (P. E. A. Teal, unpublished) support the idea that the ability to respond to pheromone is not hindered by exposure to some neurotoxins. In our experiments, we loaded rubber septum lures with both 2 mg of (±)-disparlure, the pheromone of the gypsy moth, and up to 20 mg of either fluvalinate or dichlorvos. Both field trapping and flight tunnel studies indicated no differences in taxis or landing of male gypsy moths between pheromone–pesticide lures and the pheromone lures alone, although all males tested in the flight tunnel died within 24 hr after contacting the pesticide lure (Teal, 1986). If the effect of these pesticides were to cause dysfunction of the receptor neurons immediately on contact, then even small numbers of pesticide molecules encountered by the antennae in the pheromone plume would have disrupted behavior and certainly would have caused differences after males contacted the lure. However, the number of moths reorienting to the lures and subsequently landing a second time was no different for lures containing pesticide plus pheromone or pheromone alone. While the results of our flight tunnel study are preliminary they do support the hypothesis expressed by Linn and Roelofs (1984) that neuroactive substances function on the central rather than peripheral nervous systems.

Still other studies are being conducted using inhibitors for sensory disruption. Compounds that are structural analogs of pheromone molecules can be used to block pheromone receptors (Prestwich, Chapter 14, this volume). For example, the acyl fluoride mimic of (Z)-9-tetradecenal has been shown to change dramatically the response of tobacco budworm moths to their pheromone. These studies will facilitate the development of an understanding of the role of macromolecular pheromone receptors in semiochemical-medicated communication.

IV BIOCHEMISTRY OF PHEROMONE SYSTEMS

While research in the areas of pheromone isolation and identification and the behavioral aspects of pheromone communication has been extensive over the past 25 years, studies on pheromone biosynthesis and the biochemistry associated with pheromone perception are relatively new. The biochemical investigations build on and complement work in the areas of pheromone identification and behavioral analysis, and they are thus important in accurately defining pheromone communication systems.

Pheromone biosynthesis determines the compounds that will be produced and emitted as pheromones and also establishes the ratio of components in the blend that is released. This is demonstrated by the pheromone blends used for communication by members of the Spodoptera genus of noctuid moths. The southern armyworm, S. eridania (Cramer), uses a series of four mono- and diunsaturated 14-carbon acetates and a monounsaturated 16-carbon acetate for communication (Teal et al., 1985). Beet armyworm moths, S. exigua (Hübner), do not respond to this blend of acetates in the field (Teal et al., 1985) but are attracted when (Z)-9-tetradecenol, a major component of its pheromone (Tumlinson et al., 1981), is included in the blend. Interestingly, this alcohol is present in pheromone gland extracts of the southern armyworm but is not released, suggesting a close biosynthetic relationship between alcohols and acetates among members of this genus.

Gene expression is ultimately responsible for establishing pheromone communication systems. While the majority of species produce and respond to a reasonably constant blend of pheromone components, there are examples where isolated subgroups of a species have modified the biosynthetic pathways and produce altered blends. Miller and Roelofs (1980) have demonstrated that a laboratory strain of the redbanded leafroller moth has a significantly lower proportion of (E)-11-tetradecenyl acetate than does the feral population in New York. While this situation is artificial and may reflect selective pressures due to laboratory rearing, cases have been documented in which feral populations produce and respond to different blends. For example, two pheromone races of the European corn borer, Ostrinia nubilalis (Hübner), exist in North America, having been imported from different areas of Europe. The pheromone used by this species is a blend of (E) and (Z)-11-tetradecenyl acetates, but apparently during a period of geographic isolation two pheromonally distinct races of moths evolved. One strain uses a 96 : 4 E : Z blend while the other responds to a 3 : 97 E : Z ratio of the acetates (Klun et al., 1973; Kochansky et al., 1975). In areas of North America where the two races exist in sympatry, evidence indicates that males respond preferentially to the pheromone of their own race and that the races are not interbreeding freely (Cardé et al., 1978). Studies by Klun and Maini (1979) on hybrid and backcross individuals of the two strains have indicated that both pheromone blend production and perception are controlled by the simple autosomal inheritance of a single pair of alleles. However, the F1 hybrids of both parental crosses contained a 65 : 35 E : Z ratio of the acetates rather than the expected 1 : 1 blend. Klun and Maini (1979) explain the disparate ratio in terms of incomplete dominance by the allele governing the E isomer of the acetate.

A similar case has been documented for eastern and western strains of I. pini. In this case, the western population produces and responds preferentially to (–)-ipsdienol while the eastern race uses a 65 : 35 blend of both enantiomers (Lanier et al., 1980). Both races preferentially respond to their own pheromone (Lanier et al., 1972), and hybrids of the two populations respond maximally to the hybrid pheromone blend (Lanier et al., 1980). As in the case of the European corn borer, the genetics of both pheromone production and attraction are governed by autosomal genes.

Studies on pheromone biosynthesis are useful in defining the pheromone blends produced by insects and in establishing the biosynthetic mechanisms responsible for maintaining pheromonally regulated reproductive isolation. Furthermore, by comparing the biosynthetic pathways used by males and females for pheromone production we can obtain valuable information regarding the molecular genetics of pheromone production. Recent studies conducted in our laboratory on the terminal steps in pheromone biosynthesis in both males and females of a number of species of Heliothis moths serve to illustrate these features. Females of H. virescens release a volatile sex pheromone blend composed of tetradecanal, (Z)-9-tetradecenal, hexadecanal, and (Z)-7-, (Z)-9-, and (Z)-11-hexadecenals (Pope et al., 1982; Teal et al., 1986), but the corresponding alcohols are also present in the pheromone gland in approximately the same ratio as the aldehydes (Teal et al., 1986). The major alcohol component, (Z)-11-hexadecen-1-ol, has no apparent effect on males of this species (Vetter and Baker, 1983), and while it is present in the pheromone glands of two other species, H. subflexa (Teal et al., 1981; Klun et al., 1982) and H. zea (Teal et al., 1984), inclusion of it in pheromone lures of these species results in decreased capture of males.

In vivo application of hexadecan-1-ol and other primary alcohols to the surface of the pheromone gland of H. virescens indicated that the alcohols were rapidly converted to the corresponding aldehydes (Fig. 2). Secondary alcohols were not converted to the corresponding ketones. Conversion of the alcohols to aldehydes was inhibited when preparations were treated with the primary alcohol substrates under nitrogen. However, the conversion proceeded when air was substituted for N2 after 15 min (Teal and Tumlinson, 1986). These results demonstrated that the enzyme responsible for the alcohol to aldehyde conversion was an alcohol oxidase specific for primary alcohols. Further, the production of a 1 : 3.5 ratio of (Z)-9-tetradecenal : hexadecanal when a 1 : 3 ratio of the corresponding alcohols were applied, coupled with the similar ratios of alcohol precursors and aldehyde pheromone components found in gland extracts (Teal et al., 1986), demonstrated that substrate-level competition for the enzyme by the alcohol precursors regulates the blend of aldehydes produced as pheromone components. A similar biosynthetic system which utilizes an alcohol oxidase has been identified for the spruce budworm moth (Morse and Meighen, 1984, 1986 and Chapter 4, this volume), and suggests that oxidases may be common among Lepidoptera species that produce aldehyde pheromone components.

Fig. 2 Chromatograms of gland extracts of Heliothis virescens on a 30 m × 0.25 mm fused silica capillary column coated with methyl silicone. (A) Extract of untreated gland. (B) Extract of gland treated with 1 µg of 16 : OH in DMSO. (C) Extract of gland treated with a 3 : 1 ratio of 16 : OH and Z9-14 : OH.

Subsequent studies (Teal and Tumlinson, 1987) established that the conversion of the alcohols to aldehydes occurred within and on the surface of the cuticle overlying the sex pheromone gland while the alcohols were present in the gland cells alone. This is the first instance in which the insect cuticle has been demonstrated to contain enzymes involved in pheromone biosynthesis and supports Percy-Cunningham’s hypothesis that the cuticle is an integral part of the sex pheromone gland (see Percy-Cunningham and MacDonald, Chapter 2, this volume). Studies on both H. zea and H. subflexa have shown that a similar enzymatic conversion occurs in aldehyde production by these species (Teal and Tumlinson, 1986, 1987). In fact, in the laboratory we have overcome pheromone-mediated reproductive isolation between H. zea and H. virescens by inducing H. zea females to produce the same blend used by H. virescens. Appropriate amounts of the two 14-carbon alcohols that correspond to the aldehydes lacking from the H. zea blend were applied to the pheromone gland of H. zea females (Teal and Tumlinson, 1986). Female H. zea treated in this manner were attractive to H. virescens males, and repeated interspecific copulation attempts were recorded in laboratory flight tunnel studies.

The sex pheromone used by H. subflexa differs from the above species in that, in addition to the alcohols and aldehydes, the sex pheromone gland also contains the corresponding acetates in a ratio that approximates that of the aldehydes (Teal et al., 1981; Klun et al., 1982). The acetates are important for maximizing the behavioral response of males to pheromone lures, and as such we anticipated that the alcohols were precursors of both the acetate and aldehyde components. However, in vivo application studies showed that the acetate was, in fact, the precursor of the alcohol that was then rapidly converted to the aldehyde. Our present hypothesis is that the major portion of the acetate is converted to the analogous alcohol within the pheromone gland cells and that both the acetate and the alcohol are secreted into the cuticle. Thus, the pheromone released from the surface is composed of acetates and aldehydes. Strangely, the pheromone gland of H. virescens females also contains enzymes capable of converting acetates to the corresponding alcohols (Ding and Prestwich, 1986; Teal and Tumlinson, 1987) even though no acetates are present in the gland of this species. The presence of this enzyme in the H. virescens pheromone gland shows the close phylogenetic relationship between H. subflexa and H. virescens.

The similarities in pheromone biosynthesis between H. virescens and H. subflexa become clearer when considering the pheromone blend produced by the hairpencil glands of males of H. virescens ). In addition, each male equivalent of these samples contained an average of 14.2 ng of octadecyl acetate and 7.5 and 6.3 ng of the corresponding alcohol and acid, respectively. Gas chromatographic analysis of these extracts on both polar and apolar capillary columns also revealed the presence of a number of other compounds as indicated in Table I. However, adequate mass spectra have not been obtained to confirm the identities of these compounds (Teal and Tumlinson, 1987).

TABLE I

Compounds Present in Hairpencil Gland Extracts of Males of H. virescens based on Gas Chromatographic Analysis (n = 20)

Assuming that our assignments for all of these components are correct, then, in addition to the 18-carbon compounds, acetates corresponding to all of the aldehydic pheromone components released by females of H. virescens are present in the hairpencil pheromone produced by males. However, as indicated in Table I, the acetates were always present in much greater amounts than either the corresponding acids or alcohols, and the saturated compounds were present in much greater concentration than the monounsaturates of equal chain length. The latter feature suggests that although both sexes of H. virescens possess enzymes involved in desaturation (see Bjostad and Roelofs, 1983), the activities of these enzymes are regulated differently by each sex. Topical application studies indicated that the acetates are the immediate precursors of the alcohol components. Esterases capable of the same conversion of acetates to alcohols play an integral role in pheromone biosynthesis by females of H. subflexa (Teal and Tumlinson, 1987) and, as indicated above, are also present in the pheromone gland of females of H. virescens. The presence of the corresponding acids as volatile components is somewhat of an enigma. However, conversion of the alcohol to the aldehyde with immediate and complete conversion to the acid (Ding and Prestwich, 1986) might explain both the presence of the acids and absence of the aldehydes.

Thus, within the members of the genus Heliothis we see a common biosynthetic theme of pheromone production. Variations on the theme that result in the production of specific blends can be attributed to the evolution of genes responsible for the production of specific enzymes and to genetically controlled specific enzyme activities.

As indicated earlier, a complete understanding of semiochemically mediated communication systems should lead to development of more effective methods of pest management using pheromones. Continued studies on the actual blends of components released by calling insects will allow the development of lures that are fully competitive with females, thereby increasing the effectiveness of mating disruption programs. Similarly, studies on the use of inhibitors may lead to control techniques based on inhibiting responses to pheromones. Finally, knowledge of the biochemistry and enzyme systems involved in pheromone biosynthesis may lead to the development of control programs based on enzyme or hormone inhibition or may allow for the development of control programs based on genetically altered insects. Thus, while studies on the biosynthesis and catabolism of pheromones are just beginning, the field promises to be one of the most exciting areas of study for the future from both the purely scientific and practical standpoints.

Knowledge of the biochemistry of putative pheromone receptor proteins and the associated neurophysiological responses is limited, and it is important that we increase our knowledge in this area. This will lead to the elucidation of the mechanisms of perception and central nervous system processing that could lead ultimately to the development of new methods of insect control. These subjects are covered in detail by Prestwich (Chapter 14, this volume) and by Vogt (Chapter 12). It would not be surprising to find that a close link between pheromone biosynthesis and catabolism exists.

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