edited bu
products of chemistry
George B. Kauffman Calltomla State Univenlty, Fresno Fresno. CA 93740
Using Chemicals to Communicate William C. Agosta The Rockefeller University, New York, NY 10021 serve as pheromones in various situations. The only way to Our information about the outside world comes to us find out what wmpounds are carrying a message is to isolargely through sight and sound. For many other living late and identify them. creatures, the world is a much different place. Their predominant source of information concerning the world The First Structure of a Pheromone about them is chemical signals that come from other orThe first effort to identify a chemical signal was that ganisms or events in the environment. It is difficultfor us begun in the late 1930's by the German organic chemist to appreciate the broad significance of chemical signals for and 1939 Nobel laureate, Adolf Butenandt. His research other creatures because we ourselves are relatively insenlasted 20 years and led fmally to the identification of the sitive to chemicals. There are, for example, many people sex attractant of the female silkworm moth (Bombyx mori) who lack a sense of smell and still live quite normal lives. (3). During most of this time, gas chromatography did not In contrast, the ability to sense and respond to chemicals yet exist, and spectroscopic methods were primitive. is vital for species as diverse as seaweeds, boll weevils, and Butenandt and his co-workers had to devise methods for house mice. Even bacteria respond to certain chemicals, isolating the attractant from thousands of female moths moving toward amino acids and away from toxic comand then purifying and derivatizing milligram amounts of pounds. material. From half a million female moths they finally obOne important group of these chemical signals is pherotained 6.4 mg of the purified pheromone. They derivatized mones. which are the sienals that members of a species use this material to form its ester with 4'-nitroazobenzene-4to communicate with o>e another. (The word dheromone carboxylic acid (Fig. I), because they could handle the was created ( 1 )from two Greek words. herein, to transfer, and horrnon, to excite.) These signals c&ry messages such as "make more testosterone," "come over here," and "I am a receptive female" (2). For many of the species that have been studied we are aware of only a single pheromone, but social insects such as ants and honeybees probably use 20 or 30 different chemical signals to regulate their wmplex lives. Our understanding of pheromones began with late 19th-century experiments onlepidoptera. Several decades of research demonstrated that female moths and butterflies attract the male of their species, oRen over distances Figure 1. 4'-Ntroazobenzene-4-carboxylic acid. of several kilometers. By the late 1930's investigators agreed that this attraction was due to small amounts of bright red crystallme ester more readily than the colorless, volatile chemical compounds emitted by the female and deoily pheromone. Fortunately, the structure was simple tected by the male in his antennae. enough so t h a t they were able to identify this small At that time nothing was known about what chemical amount of material by degradation and total synthesis. compounds these might be. Thanks to the efforts of many The pheromone, which they named bombykol, was chemists working with many kinds of pheromones since (10E,12Z)-hexadecadien-1-01 (Fig. 2). then, we now recognize the great chemical diversity of her om ones. Totally unrelated molecules bear &litatively simila;messages in different organisms, and there are no obvious rules about the structures used by any organism. Pheromones that travel through the air need to be volatile, ones that are released in the ocean ought to be reasonably stable in water, and ones that must remain in one place should be persistent. Beyond such wmmon-sense requirements, however, there is no simple way of predicting the sort of molecule a particular species may use for carrying a mes- Figure 2. (IOE, 124-Hexadecadien-7-01(bombykol). sage. Molecules from carbon dioxide to proteins Bombykol aroused immediate interest in learning the chemical structures of other pheromones. Here was a new area of research that combined chemistry, biology, and beMuch of this article is based on material taken from the author's havioral studies. Attention soon widened from insects to book, ChemicalCommunication: The Language ofPheromones; Scientific American Library: New York, NY, 1992. other organisms up and do* the evolutionary scale. With
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time, improved techniaues for such investigations emerged and became widely familiar. The expl&ations that began with bombykol have evolved into our prcscnt store of information on pheromonal chemistry in species from mlcrobcs to mammals. Chemists and hioloj$sw: have studied many hundreds of compounds, messages, and orgunisms over the past 30 years. Perhaps the following examples will b~vesome idea of the wide variety ot'chemical structures and messages discovered in this research and also point out some of the problems enwuntered. The Sex Attractant of a Water Mold One of the simplest creatures with a n attractant pheromone that has been studied both chemically and biologically is the water mold Allomyces, a microorganism that grows on plant or animal debris (4). Like other water molds, the life cycle ofAllomyces involves both sexual and asexual reproduction. In one phase of its existence it pmduces male and female reproductive cells, or gametes, that are released into the water, where they move about, each powered by the beating motion of a taillike flagellum. For a new generation ofAllomyces to arise, it is essential that a male and female gamete find each other and fuse. To facilitate their meeting, each female gamete emits an attractant for several hours after its release from the mold. This pheromone, known as sirenin (Fig. 3), is borne away
Figure 3. Sirenin from the female gamete by water currents and molecular motion. As the molecules travel, they move farther away from one another, establishing a concentration gradient that points the way back to the female gamete. A male gamete picks up this molecular signal some distance away. I t can sense differences in the concentration of the pheromone, and it swims up the concentration gradient toward the source, eventually fusing with the female cell. Chemists and hiolo&sts were interested in isolating and s i r e ~ nund , for this thev needed an eftective blopurifying . . assay. hat is, a chemist needs s'me way to follow the isolation procedures and to know that purification is taking place. In this particular case, the male gametes swim toward the source of sirenin, and the problem was to turn this behavior into a useful semiquantitative measure of the pheromone. Biologists solved this problem in the follo&g way. Two solut~onsin a test chamber are separated by a semipermeable membrane. On one side is the test so1;tion wniaining the pheromone, and on the other is water carrying a suspension of male gametes. The gametes are attracted by the pheromone, which diffusesslowly through the membrane. They swim up the madient to the membrane and attach themselves to it inproportion to the concentration of sirenin on the other side. Counting the number of male gametes attracted to a unit area of membrane furnishes a measure of the concentration of sirenin in the test solution. Using this bioassay as a guide, chemists were able to isolate and ourifv enoueh sirenin to determine its structure and to fAd th"at it egectively attracts male ga-
Figure 4. 2-Carene metes a t a concentration of 24 picograms (1pg = 10-l2 g) per milliliter of solution. Sirenin turned out to be a sesquiterpene diol formally related to 2-carene (Fig. 4) (5).Total synthesis (6)confirmed its structure and also furnished related compounds that were tested for their ability to attract the male gametes. None of these other compounds was effective. Even small changes in the structure of sirenin render the compound ineffective as a pheromone. This structural specificity of chemical signals is not unusual. A Sea Anemone Sends an Alarm
Anthopleura elegantissima is a common sea anemone about 2 cm in diameter that mows on intertidal rocks along the California coast. As adults, many sea anemones are brightly wlored, and they grow like lovely flowers in coastal waters throughout the world. Structurally, they consist of a vertical cylinder, closed a t one end by a base that fastens the anemone to a rock. The upper end of the cylinder forms a mouth, which is surrounded by tentacles. When an anemone is feeding, its mouth isopenand visible, and the tentacles wave about. Thcy locate hod and force it into the mouth. If an anemone is wounded by a predator, it produces a n alarm pheromone that warns neighboring anemones of the daneer. An anemone that receives this signal quickly protects itself. It shakes its tentacles, pulls them into its mouth eavitv. and closes its mouth. all in less than three seconds. ~ h &the alarm signal i i n o longer present, the anemone opens and resumes its normal activity in about two hours. Investigators a t Stanford University studied the chemistry and biology of this pheromone (7). They brought anemones into the laboratory and set them in groups, one group after another, in a stream of water. If they wounded an anemone a t the upper end of the stream. the anemones in the gmup nearesi-the wounded animal contracted first, and then each woup .downstream wntracted as the flowine water carrying the pheromone reached it. This reaction provided a sensitive bioassay for the pheromone, and with its help the investigators fractionated an extract ofhomogenized sea anemones and isolated colorless needlelike crystals. Spectroscopic studies yielded the structure of this compound, which was named anthopleurine (Fig. 5). This is an exceptional pheromone in that it is a n ionic com-
Figure 5. Anthopleurine (as the chloride.) Volume 71 Number 3 March 1994
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pound. The cation is the active signal, and it triggers an alarm resuonse at a wncentration of 74 udmL of seawater. . .. Anthoplaurn alegantissimo livcsin turbulent waters that raoidlv disoeme and dilute anthooleurine. This limits the effecthene8s of the pheromone, despite its high biological activity. In practice it can alert other anemones to danger only if they are nearby. However, curiously enough, one of the anemone's most persistent predators carries the pheromone and thus warns other anemones of the predator's presence. This predator is a carnivorous marine slug (Aeolidiapapillosa) about 2- or 3-cm long. Its favorite food is the anemone, and the two species often live together. Anthopleurine is present throughout the hody of the anemone, and when the sea slug nibbles on the anemone, it ineests the uheromone alone with its meal. The sea slue does ;lot metaiolize anthople;rine readily but stores it& its body and then releases it slowly into the water. For a t least five days after feeding on an anemone, the slug sets off the alarm reaction in anemones whenever it approaches them. On eating a bit of anemone, the sea slug effectivelyturns itself into an early-warning device. Stimulating Production of Milt in Goldfish In several animals pheromones associated with reproduction are steroidal sex hormones or wmuounds closely related to them. Scientists at the ~niversit; ofAlherta ai Edmonton have carried out thorough investigations of such a steroidal pheromone in the common goldfish (Carossius ourolus~,where a signal from the female influences the reornductive ohvsiolow of the male 18). To aooreciate this wfork, we m;st"first say a bit about ;epro&ction in goldfish. After ovulation, the female holds her eggs within her hody for several hours. These females are said to be ripe and are ready to spawn or release their eggs. A ripe female spawns with several males chasing and competing for her. She releases her eees ... . "iust as one or more of the attendant males discharge milt isperm and seminal fluid) so that the eees are fertihzed in the water. The adult fishes ignore the eggs and do not care for their progeny. Like other fishes with similar reproductive habits, female goldfish produce great numbers of eggs. Commercial breeders strip eggs or milt manually from a goldfish hy applying slight pressure to the abdomen while holding the fish. They mix eggs and milt and then pour the mixture into an aquarium. In this way they can get 10,000 fertilized eggs from a single female in one operation. When a female goldfish is nearing ovulation, the wncentration of the steroidal sex hormone 17a,20R-dihydroxy-4pregnen-3-one (Fig. 6) increases in her blood and stimulates final maturation of her eggs. As the hormone's concentration builds up, she releases it into the water, where it diffises away and reaches male goldfish.The discharged hormone now acts as a pheromone, and the male responds to this signal with a dramatic increase in milt formation. Males exposed overnight to the steroid yield over five times as much milt as males that receive no pher-
omone. This chemical signal sent by the ripe female impmves her chances of having her eggs fertilized and improves the male's chances of passing on his genes. Often many males discharge milt near one spawning female so a male's volume of milt is probably important to his reproductive success. The discovery of the pheromonal action of this stemid was an accident. The Alberta bioloeists iniected the comwund into male goldGsh to detcrmine;ts effed on milt prod;ction. Milt increased in the iniected males and also in wntrol males held in the same tank: even though they had not received injections. The investigators eventually found that a minute amount of the steroid in the tank water, presumably released by the injected males, induced this response in the wntrol males. The oheromone is effective even a t a wncentration of ahuut 50 femtograms ( 1 fg = 10-'"1 per milliliter, reflecting the maleli extraordinadv . high wnsitivitv to this stemid. Here one compound serves as both hormone and uhemmone in a sin& species. When the word was coined to designate intrasuecific chemical sienals. a distinction was intentionally made between phekmones and hormones ( 1 1 .Pheromones were dischar~edexternally into the environment, and hormones were secreted internally within an organism. We now know that this distinction can break down. Steroids exist throughout the animal kingdom and have well-recognized functions as hormones. With this in mind. it seems natural to reeard 17a.20R-dihydroxy-4-pregne~-3-onein goldfish as a gormone that became adapted to act as a pheromone. Discharge of the steroid into the water a t the appropriate time would occur as a consequence of its use as a hormone by female goldfish. Then, sensitive receptors for the wmpound and a suitable phvsioloeical response to it would eraduallv evolve in the kale and permit^the excreted hormone to acquire an auxiliary role as a uheromone. A number of hormones have probBbly taken on roles as pheromones in this way. It is also likely that, in the reverse process, some pheromones have evolved into hormones. -
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Pheromones in Mammals Many mammals are nocturnal and have a h i ~ h l vdeveloped sense of smell so it is not surprising that tGefdepend on chemical communication. In fact, their complex lives require many sorts of chemical messages. These messages may disclose the reproductive status of a female or permit the recognition of individuals. They may determine the onset of puberty in immature females or influence the timing of an estrous cycle. Other signals warn of danger or cause a male to mount a receptive female (9). Our understanding of mammalian pheromones is limited, owing lareelv to the complexitv of mammalian life andthe m&&& il brain. M k a i s integrate information received by their various senses, and this enormouslv complicates efforts to understand any specific signal. 1; addition, interpreting the reaction to a signal can he difficult. The reactions of mammals are not automatic, and their behavior is not necessarily reproducible. Sometimes a uheromone oroduces no obvious resuonse in a mammal. p&haps the a'nimal has ignored the signal, or perhaps it has learned somethine for future use. Desuite these uroblems, we now know something about the chemistry of H few mammalian uheromones. Deer. hamsters. mice...uies. - . and a few other species have received considkrable attention from both bioloeists and chemists. In wneral. behavioral studies on mammalian pheromones are much more extensive than chemical research. ~
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Figure 6. 17a,20fl-Dihydroxy-4-pregnen-3-one 244
Journal of Chemical Education
Provoking Aggression in House Mice The pheromones i n t h e urine of house mice (Mus musculus) were the first mammalian signals subjected to
extensive examination. One of the signals in male mouse urine provokes aggression in other males, and a team at Indiana University has studied the chemistry of this signal in considerable detail (10). Male mice can be taught to fight, and when challenged with a normal male, a fighting male sniffs and then starts to bite and chase the other mouse. Fighting males pay little attention to castrated males, indicating that the pheromone is absent from their urine. Comparison of the urine of normal and castrated males by gas chromatography showed that the concentrations of two components, 2-sec-butyldihydrothiazole(Fig.
Figure 7. 2-secButyldihydrothiazole
area concerns insect pheromones. One reason for this attention to insects is their economic importance as agricultural, health, and forestry pests. We wish to restrain destruction of croos hv insects. but we Dav a high price in undesirable en&o&nental e h t s for ;hi continue'd heavy use of insecticides. For this reason, the possibility of controlling pests with natural chemical signals is clearly attractive. In principle, this could mean deployment of nontoxic substances that affect only specifictarget organisms. In practice. oheromones have found several applications in contrbl; but their widespread use in agri<ure is not yet a reality (12). The currently most important practical application of pheromonel; is their use as a tool fo-r estimatiniinsect populations. Many ofthe significant agricultural pestsare larval butterflies or moths, and the attractant pheromones released by the adult forms ol'hundreds of'these pests have been identilied. Traps baited with the attractant of a particular pest are set out, and from the numbers of individuals lured and caueht. enromoloeists can estimate the total local population $the insect. ~ ' b n i t o r i a n pest ~ population in this wav after s ~ r a v i n ewith insecticides permits farmers and scientists io eva1;ate the success of the operation. Monitoring also serves as a critical warning system. Governmental agencies routinely follow local populations of several exotlc insect pests with pheromonc-balled traps, particularly in l.'lorida, Texas, and California. Another use of pheromones in pest control is a n extension of the idea of t r a o ~ i n ea reoresentative samole. With enough traps, perhaps all, or all of one sex, of a pest species could be trapped and its life cycle broken. There have been extensive tests of this "trap-out" idea, most notably in Norway and Sweden following an explosion of spruce bark beetles (Ips typographus). Traps captured nearly three thousand million beetles the first year they were deployed and nearly four thousand million the next year. Even though the tram eliminated these hupe numbers of beetles, there was a devastating loss of trees. Whether the trapping pro-gram prevented even worse destruction is uncertain. A third application ofchemical signals, known as mating dlsruption, is similar in concept to radiojamming, in which a oowerful interfering is broadcast to block receo" simmal " tion on particular wavelengths. In the same way, a n attractant t her om one soread throu~houta crop area should insect to ibcate prospective mates make it iifficult for that are emitting the same pheromone. Insects typically respond to their attractant by flying upwind within the s ~ r e a d i n eairborne sienal. Ordinarilv. ".this behavior leads to a mate. However, if there are also many artificial sources of the pheromone in the same area,. flvinn . -upwind . should lead oniy to confusion. Because pheromones are so soecific and function in such low concentrations, the idea okmating dlsruption has long hren an appealing approach to pest control. In field applications, the disrupting pheromine is dispersed from a n airplane, or alt&maii;ely i t evaporates from dispensers scattered throughout the area. 1f dispensers are used, many insects are attracted to them, and the technique resembles the trap-out strategy. Amodification of mating disruption makes use of dispensers containing both an insecticide and a pheromone, a mixture known as an "attracticide". The pheromone intederes with mate location, and the insecticicie kills many of the insects outright. One of the undesirable side effects of spraying with insecticides is the wholesale destruction of both beneficial insects as well as pests. The attracticide technique permits use of an insecticide to eliminate a pest without indiscriminately eradicating useful species. Attempts to trap out insect pests on a small scale sometimes leads to practical difficulties A small number of traps in a garden can draw many more insects than they .s
Figure 8.Dehydro-exo-brevicomin.
7)and dehydm-em-brevicomin (Fig. 81, differed greatly in the two samples. These compounds were prominent in normal urine but virtually absent from castrate urine. When both comoounds were added to castrate urine, i t was transformed into a signal that provoked aggression in fighting males. If only one of the two compounds was added to castrate urine, it remained inactive. The combination of these two comoounds a ~ ~ e a r ea td first to be the pheromone, but t h e ~ n d i a n aehemists uuicklv learned otherwise. If thev dissolved the two comin water rather than in eastrate urine, there was no activity This and other results indicate that both these compounds are indeed required, but that they have no oheromonal activity by themselves. Neither these two compounds alone nbr castrate urine lacking these compounds can trigger aggression. Here the complex odor of many, or perhaps all, of the volatile components of mouse urine, specifically including the odor of these two compounds, is necessary to elicit aggressive behavior. There are many other examples of complex odors serving a s mammalian her om ones. In some cases removal of one specific compound reduces or abolishes pheromonal activitv. In others. no single comoound seems to be critical, but compound or group of compounds reduees removal of activity. An important problem in current chemical research on pheromones is how to define and distinguish such complex signals (11). A Place for Pheromones in Pest Control Discussions of pheromones often deal only with chemical signals in insects, because most of what we know in this
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the pests. If the pheromone works well in practice, gardeners will have a new means of fighting foraging insect pests without resorting to broad-spectrum insecticides. What of the Future? We have learned a great deal about pheromones in the ~ a s30 t vears. and techniaues are in lace that should Dermit us to discover and study new pheromones analogous to known ones for decades into the future. To do more than that and expand this research beyond current capabilities, we must solve a number of problems. Among other things. we must learn how to deal kith complex od&s and how-to handle and identify organic compounds l~elowthe picomam level. We must hettrr understand olfaction and other areas of neurobiology to find out what happens between an oreanism's rece~tionof a chemical sienal and its resoonse heto%. Broad pro61ems of this sort ha; implications vond her om ones. and thev alreadv receive the attention bf m k y chemists' and bioiogists. J(s they are solved, research on pheromones can expand in novel ways and engage first-rate scientific effort for years to come.
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Figure 9. a-Terpineol.
fk
CH3CH2CH2, H/c=c
/H \ CHO
Literature Cited Figure 10. (E)-BHexenal(leafaldehyde). can hold or kill. Pests continue to arrive from afar long after the traps are full, and the unfortunate consequence is to increase the number of voracious pests in the area rather than to destrov them. One wav of usine her om ones that avoids this probiem is to disperse a signii that brings s canbeneficial insects to the earden rather than ~ e s tthat not be destroyed. ~ l u r e T h adoes t just this is now commercially available after several years of development by scientists a t the US Department of Agriculture. Instead of attractingpests that eat garden plants, this pheromone attracts spined soldier bugs (Podisus maculiuentris), which are carnivorous insects that eat pests. The pheromone is largely a mixture of a-terpineol (Fig. 9)and (El-2-hexenal (leaf aldehyde) (Fig. 101,and it is the signal broadcast by male soldier bugs to attract females (13).Acommercial dispenser of this pheromone in a garden infested with insect predators should bring female soldier bugs eager to devour
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Journal of Chemical Education
lando, 1987. 3. Butensndt. A,: Beekmann, R.:Stamm, D.; Hecker, E. Z Nolurforfoh. B 1958, 14, 283: Heeker, E.;Butenandt,A. In Teehnigupr inPhemmamRe~e~emh: Hummel, H. E.; M i l k T. E., Eds: springer:New YWk, 1984:Chapter 1,pp 1 4 4 4. PommerviUe, J. T h e Role of Sexual Pheromones in Allomyas" in Samal Intame*lons in ~ ~ k~ f ~~ ~ .bonav. ~ e s t: D. H.: i . H ~O "X ~.. P A .. E ~ s .. :~ ~pros=: ~ N~WYO mi. ~~, 5. Machlis, L.; Nutting, W. H.;WiUiams, M. W.; Rapport, H. Bimhem. 1953.5.2147; Machlis, ;.I Nutting, W H.; R a p p m t , H. J Am. C h e n Soc. 1968,90,1674. 6. Rapoprt. H.; Piattner. J . J . J . A m Chem S o c 1971.93, 1758. 7. Howe, N. R.: Sheikh.Y.M.Scienn 1975,189,386. 8. Sorenaen, P W: Staey, N. E. In Chemiml Signals i n Mrtebmtes; MacDonald. D.: MuUerSehwane, D.: Natynezuk, S. E.. Edr.: Oxford University Resa: Odor& 1990:Vol V, pp 302-311, and references cited therein; Stacey, N.; Sorensen, P. In Bimhemrstryond Mohculor Bioiogy offihes: Hochachka, P W: Mommsen,T. P., Eds.; Elmvier: Amsterdam, 1991: Vol. I, Chapter 5. 9. Albone, E. S. Mammalian SPmioehemistry: John Wiley: Chicheater, 1984. 10. Novotny,M.;Harvey S.;Jemiolo,B.;Albcrts.J P r o c N a f l . Acad Sci. USA1986,82.
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0"CO
11. Smith, A. B., 111; Belche~A. M.; Epple, G.; Jars, P C.; Lsvine, B. Scionn 1885,228, 175; Belcher, A. M.; Smith. A B., 111; Jurs, P. C.; lanne, B.; Epple, C.J . Chem. E d . 1986,Z2,513. 12. Ridgeway, R. L.;Silverstel", R. M.:Insme, M. N. Behauior-modifiing Chemimls for 1-d Monog.ment; Marcel Dekker: New Yo* 1990. 13. Aldrich, J. R.;Koehansky J.P.:Abram&C. B.Enuiron.Enfomd. 1984,13,1031.
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