Chemical Communication

The Language Of Pheromones

Agosta, William C.

Henry Holt and Co.

Chemical Communication
A female silkworm moth, perched on a cocoon, emits a chemical signal that attracts a mate. So small is the amount of chemical produced that to isolate and identify the substance scientists needed twenty years and over half a million female moths.
Human beings gather information about the outside world largely through sight and sound. These are our best-developed senses, and when we want to know what is going on we look and listen. For many other living creatures, the world is a much different place: for them chemical signals are the primary source of information. Chemical compounds from other organisms or events in the environment provide their basic knowledge of the world. Even many creatures that do have other well-developed senses find chemical signals to be indispensableSpecies as diverse as water molds and elephants depend on the ability to sense chemicals in the world around them for survival. Even simple bacteria respond efficiently to certain chemicals, moving toward foodstuffs and away from toxic compounds. Humans must have developed an early appreciation of chemical signals. The attraction of a male dog to a female, for example, could have provided important clues thousands of years ago. Even earlier, hunters must have learned about the significance of scent to both prey and predator. However, our organized, scientific understanding of chemical signals is a much more recent development. This understanding began in the nineteenth century with the experiments of naturalists who were intrigued by the ability of female moths and butterflies to attract the male. For decades these investigators documented attraction for various species, often over distances of several kilometers. Through clever experiments they demonstrated that functional antennae were critical for the male to locate the female: males could no longer find females when their antennae were covered with lacquer or totally removed.
These observations were fascinating, but scientists achieved a satisfactory interpretation of them only slowly. Initial explanations invoked some sort of radiation from the female, and one idea was that the male's antennae were appropriately tuned to receive the wavelength broadcast by the females of his species. As late as 1950, investigators found that infrared radiation emanated from the region of the thorax of females of two species of moth. In their report of this discovery, these scientists wondered if this radiation might be the signal emitted by the female to lure her mate.
On a more rewarding track, as early as the 1930s there were reports of the extraction of "attractivity" from female moths. There was also evidence that attractive substances were present in the air over cages containing females. Experiments eventually showed convincingly that attractivity must be due to volatile chemical substances. At the end of the 1930s this conclusion was firmly enough established that the German chemist and Nobel laureate Adolph Butenandt became interested in undertaking the isolation and identificationof such a sex attractant. After some twenty years this research led to the identification of bombykol, the sex attractant of the female silkworm moth Bombyx mori. This investigation holds an important place in our understanding of pheromones, and we shall return to it later.
At about the time that Butenandt completed the work on bombykol, Peter Karlson and Martin Luscher coined the word pheromone to describe a chemical signal transmitted between members of the same species. (The word was created from two Greek words, pherein, to transfer, and hormon, to excite.) They wished to distinguish pheromones from other kinds of chemical signals that convey information to organisms. Some of these other kinds of signals come from whole systems such as rivers or forests, as when the odor of its native stream guides a salmon returning after years in the distant ocean. Other chemical signals pass from members of one species to another: dying trees release substances detected by bark beetles that then attack the trees; skunks defend themselves against other animals with an unforgettably malodorous spray. None of these examples concerns a message sent and received within a single species, and so none of these signals is a pheromone. There is also another kind of chemical signal that is not a pheromone, even though it does concern a single species. This is a signal that operates within a single individual, delivering a message from one part of the organism to another. These signals are known as hormones, and they will turn up again later in our explorations.
While Butenandt was setting out to isolate bombykol, the famous behaviorist Karl von Frisch was making one of the classic discoveries of chemical communication. An Austrian zoologist most widely known for his work with honey bees, von Frisch (1886--1982) shared in the Nobel Prize for Physiology or Medicine in 1973. His contribution to the study of pheromones emerged from his work with the European minnow Phoxinus phoxinus, and it came about through two casual observations. Thefirst concerned a minnow that von Frisch had marked with an incision near its tail. When he returned this individual to the school, the other minnows became frightened and some of them retreated. The second observation followed von Frisch's finding a minnow stuck under the rim of a feeding tube, struggling to free itself. He released the fish, and when it swam away toward the group, they all fled in fright. To von Frisch these episodes were curious enough to warrant detailed investigation, and his carefully documented results set the direction of much later research into chemical communication in fishes.
Von Frisch called the agent responsible for this fright reaction the "alarm substance." Subsequent work has shown that most members of this group of fishes (the ostariophysians) show a fright reaction when an injured ostariophysian is introduced into the water nearby; the nature of the reaction itself varies with species, sex, and location of the reacting fishes. They may swim in a tighter school, leap at the surface, swim away and hide, or sink to the bottom and become very still. In some instances they will avoid the area of their disturbing encounter for some time thereafter. In at least one species the fright reaction changes over the life of the fish: young creek chub (Semotilus atromaculatus) dart away and hide, but adults drop quietly to the bottom and remain motionless.
This sudden and remarkable reaction is under the control of a pheromone in the skin of the fish, where it is carried in large alarm-system cells. These cells are fragile, and upon injury they rupture, discharging the pheromone into the water. Simply scaring a fish does not affect its alarm-system cells, but even minor mechanical damage to the skin will disrupt them, and damage to only a small area of the skin of a single fish is capable of causing fright in an entire school.
The alarm substance and bombykol are just two of thousands of pheromones. All sorts of organisms use chemical signals to convey a rich variety of messages. The alarm substance signals "danger--get out of here!," and bombykol is the female moth's message "come to me." Other pheromones bear messages such as "the queen is in the hive and all is well," "produce more sex hormone," "we are under attack!" and "I am pregnant." Messages may elicit a specific behavioral response in the recipient, such as swimming toward the source of the signal or attacking an enemy. Alternatively,the message may cause a physiological change in the recipient, perhaps altering the timing of the sexual cycle or inducing puberty. For some species we are aware of only one pheromone and a single message. Other species, such as ants and honey bees, use many different chemical signals to coordinate the activities of their complex communities.
As we consider the significance of pheromones in the living world, it is important to keep in mind that human beings make relatively minor use of the ability to sense chemicals in the environment. Our primary means of detecting chemicals is through olfaction, the sense of smell. The sensitive discrimination of professional perfumers and wine tasters demonstrates that considerable refinement of the human olfactory ability is possible, but we are much less sensitive to chemicals than many other species. It is quite normal for some people to be a thousand-fold more sensitive than others to specific chemicals, evidence of how variable the importance of olfaction is to humans. Without a sense of smell, we would certainly find the world a much poorer place, but for us olfaction is not critical to survival or even to a reasonable existence. People who lack a sense of smell are said to suffer from anosmia. This condition accompanies several serious maladies, but there are many anosmic people who show no other defect and who live quite normal lives. For human beings, anosmia is not even remotely comparable to the losses entailed in blindness or deafness. In contrast, chemical communication plays an indispensable role in the lives of the creatures that we shall discuss in the pages that follow.
Pheromones as Chemical Compounds
Pheromones are specific chemical compounds or mixtures of compounds. This means that they are real objects that are endowed with definite physical and chemical properties. Once released, a pheromone has a physical existence quite apart from the organism that produced it. A volatile chemical signal may be carried on the breeze or in a current of water, to deliver its message at a later time and in a place remote from its source. Such a signal could be effective at attracting faraway mates or helpers. Chemical signals may also be persistent. Some of them can be deposited on a bush or on the ground, to be detected where they were left after the sender has departed. If such a signal is chemically stable and not too volatile, it may remain in place, and active for days. Stability and nonvolatility are good properties for a signal used, for example, to delineate the boundaries of a territory or to mark a food source. Conversely, the signal may be chemically unstable and destined to provide its message only briefly. This might be useful in a pheromone employed to give an immediate and short-term warning of danger.
Pheromones that travel through the air need to be volatile, ones that are released in the ocean must be stable in water, and ones that must remain in one place for an extended period should be persistent. Virtually all the compounds that will appear in the pages to follow are organic molecules. Molecules are groups of atoms assembled into a specific three-dimensional arrangement or structure, and organic simply means that these molecules contain carbon atoms. Properties such as volatility, persistence, and stability have their basis in molecular structure. The smaller a molecule of a given kind of is, the more volatile the compound will be. Two simple molecules illustrate the effect of size: pentane has 5 carbon atoms and pentadecane has 15, but otherwise they look much alike; yet the small size of the pentane molecule leads to high volatility. A small amount of pentane poured into a saucer will evaporate in a minute or so, whereas the much larger pentadecane molecules form an oily, persistent substance. A small amount does not evaporate noticeably in hours.
Volatility and persistence also reflect the functional groups present in a molecule. These are groupings of atoms that appear repeatedly in the structural formulas of organic compounds; they are attached to or embedded in a framework of carbon atoms. Several of the most common ones are illustrated below:
The double lines indicate that atoms are connected by two bonds, called a double bond. Because double bonds are flat and rigid, functional groups help determine the shape of molecules. Groups of atoms can twist about single bonds, but the two atoms bonded by a double bond remain rigidly bound.
This property of double bonds has an interesting consequence. There are two possible arrangements of two atoms attached to opposite ends of a carbon-carbon double bond, called E (from the German entgegen, opposite) and Z (from the German zusammen, together). Older names for these two arrangements are trans and cis, respectively. The two forms of 2-butene are two different chemical compounds, and each has characteristic chemical and physical properties.
The effect of functional groups on volatility is well illustrated by the two compounds propane and propanol. These two compounds differ by only one oxygen atom, but this atom is part of a hydroxyl group, and its presence has a striking effect on volatility. Propane is much more volatile than pentane, but propanol is much less so. The hydroxyl groups in different molecules of propanol interact with one another and reduce its volatility. Propanol behaves like a larger molecule than it is.
Functional groups are sites of reactivity in a molecule. They make new bonds with various other molecules and ions (atoms or groups of atoms that carry electrical charge), and the strength of their tendency to form bonds determines whether a compound is reactive or inert, stable or unstable. These are relative terms: a molecule with functional groups that react with water but not with dry air would be unstable in the ocean but could be stable in the desert. The alarm pheromone of a minnow has functional groups that make it reasonably stable in water, whereas the attractant of the golden hamster has functional groups that make it reasonably stable in the hamster's desert home.
Throughout the natural world a wide variety of molecules serve as pheromones. Totally different kinds of molecules bear qualitatively similar messages in different organisms, and there are no obvious rules about the structures used. Beyond commonsense requirements that a pheromone be suited to its environment, there is no simple way of predicting the sort of molecule a particular species may use for carrying a message. The only way to find out what compounds are acting as pheromones is to isolate and identify them chemically. This was Butenandt's goal in working with bombykol, and it remains the main chemical problem in pheromone research today.
The Scientific Study of Pheromones
In the years immediately after the success with bombykol, the study of pheromones achieved wide popularity. Our present knowledge in this area results from thousands of separate studies over the past thirty years. Pheromones are extremely widespread in nature, and scientists have studied the pheromones of animals, algae, fungi, and bacteria. Very little is known about whether plants use chemical signals, although there is one tantalizing report that points to pheromones in poplar and maple trees. Most of the current fund of knowledge comes from work on insects, followed distantly by work on mammals.
Studies of pheromones usually begin with a simple observation. That is, when presented with the putative signal, the organism under study changes physiologically or behaviorally. There are many reports that have not yet beencarried further than this. Dealing with such observations over the years has instilled caution in interpreting them, and usually the next step is to design and carry out more extensive experiments with careful controls. In favorable circumstances these experiments establish a clear connection between a signal from one organism and its reproducible effect in other members of the same species. Much of our present knowledge is at this level. Once the existence of a pheromone and its effect is firmly established, investigations may go in various directions, from group behavior to neurophysiology to genetics, with the aim of understanding the pheromone more fully from various points of view and of fitting it into a larger body of behavioral and biological knowledge. At this point there may also be an effort to identify the chemical compounds responsible for the pheromonal effect.
Identifying these compounds requires separating them from the natural mixture or secretion in which they occur. The systematic separation of a mixture of chemical compounds into its pure components is called fractionation. Chemists take advantage of differences in the chemical and physical properties of the components of a mixture to partition these components between two or more states. This partitioning may be between liquid and vapor, or solution and solid, or solution and adsorbent, among other possibilities.
A chemist who wishes to identify the active compound or compounds in a pheromone often begins with the total contents of a gland that makes and stores the pheromone, or perhaps he begins with gallons of seawater containing the pheromone along with hundreds of other components. He must have available a wide assortment of procedures for fractionation, because pheromones include compounds with many different sorts of chemical and physical properties. The chemist may require many steps and several different procedures to purify the compound or compounds responsible for the chemical signal.
Fractionation techniques can be effective only if the investigator has some means of following his progress. After performing a separation step, he must answer such questions as: Did the procedure work? In which fraction or fractions is the desired material? How much purer is the desired material now than before? An assay is essential, and ideally it should be quick, cheap, and dependent in a known fashion on some property of the compound being isolated. If the compound is a pheromone, the only property initially known to the investigator usually is its biological effect on the natural recipient. The investigator needs some sort of procedure that uses this biological effect to guide his fractionation, and this procedure is a bioassay. An obvious way to follow the isolation of a sex attractant, for example, is to gauge the attractivity of fractions by testing them on the natural recipient of the pheromone. To evaluate a sample's attractivity, the investigator will normally score or time some activity, such as movement toward the sample or the beating of an insect's wings.
Once the investigator has the pure components in hand, he can identify them. For each component, he must establish the number and position of the atoms present in its molecules. Structure determination, as this is called, relies on a combination of chemical and physical techniques. By carrying out chemical reactions and physical measurements on the compound, the chemist can obtain a complete count of all atoms present, determine the carbon framework of chains and rings, and identify and locate all functional groups.
Nearly always, the chemist working with pheromones must isolate, purify, and identify compounds that are available only in minute amounts. One of the most frequent observations in this area is that all kinds of organisms are exquisitely sensitive to their pheromones. Often only a minute amount of material is necessary to send a chemical message, even to a large number of recipients. Consequently, an organism may never keep more than a little of the pheromone on hand. A female silkworm moth ordinarily contains enough bombykol to excite many millions of males, but this requires only about 1.2 micrograms of material. Moreover, even the most skilled investigator loses material during isolation and purification, and so only very small amounts of most pheromones can be obtained for chemical study. In early years this was a technical barrier that virtually prohibited rigorous chemical identification of pheromones. Butenandt and his group had about 6 milligrams of bombykol available for its identification in the 1950s, and they were forced to devise novel methods to solve the problem. Fortunately, owing to technical improvements over the years, scientists can purify and identify ever smaller amounts of material. A present-day chemist could probably identify bombykol using less that a hundredth as much material as Butenandt needed. Some substances can now be identified from samples of only a few picograms.
With a knowledge of the chemical constitution of a pheromone, new research problems present themselves. How does the organism transform chemical compounds in its diet into the pheromone? How does the pheromone deliver its message to the recipient? Answers to such questions depend on knowing what the pheromone is, that is, on knowing the chemical structures of its components. At present, structures are available for only a small fraction of the pheromones that have been described.
An Interdisciplinary Effort
The study of pheromones is an area of scientific research that draws information, techniques, and investigators from behavior, biology, and chemistry. One of the attractions here is the opportunity to work collaboratively with scientists who are trained in other disciplines and who sometimes bring a quite different perspective to a shared research problem. Few investigators are equipped to work equally well in all these areas, and interdisciplinary cooperation can be especially fruitful.
In the pages that follow we shall sample what is known about pheromones in water molds, insects, mammals, and many other creatures, drawing largely on examples where behavior, biology, and chemistry have all contributed to our current knowledge. We cannot discuss all that is known, but we will explore many kinds of organisms and their chemical signals and also have a look at how scientists go about their research in this area. We start with simple organisms and then move through the animal kingdom to the mammals. In the final chapter we consider the provocative question of pheromones in humans.
It is certainly possible to appreciate the fascination of pheromones without a detailed knowledge of the disciplines that contribute to their understanding. A number of terms, concepts, and techniques come up repeatedly, and the glossary following Chapter 7 contains definitions and brief discussions that may clarify these matters.
Copyright © 1992 by Scientific American Library