Research at Dartmouth on Fly Control

The Chemistry Department of Dartmouth Collegeprovides the opportunity for qualified undergraduatestudents to do original and sophisticated chemical research.This affords the student a unique view of the world ofscientific research and better prepares him for an earlycareer decision. The following article by John Sanstead '72represents but a fraction of the total research that he hasdone with me since his summer freshman term. Mr.Sanstead's results, as described herein, may well revolutionizefly control.

—GORDON W. GRIBBLE Assistant Professor of Chemistry

... And the small gilded fly Does lecher in my sight. Let copulation thrive!

Shakespeare, King Lear

The common housefly, Musca domestica Linnaeus, is more than just a noisome pest that uninvitedly drops in on us on hot summer days. It is a danger to the health of man and animals, principally because it breeds in garbage, manure, and rotting crops, and, in turn, spreads typhoid, dysentery, diarrhea, cholera, yaws, trachoma, and many other diseases. Flies often deposit their larvae on the human body leading to the diseased condition of Myiasis, which is still common in America. They even serve as intermediate hosts to round worms, tapeworms, and other parasites.

The infestive nature of the fly is the result of its indiscriminate, omnivorous feeding habits. It will feed on sputum, fecal matter, and discharges from open wounds as well as on man's food. The only limitation to its feeding habits is that the food must be a liquid, or at least capable of being rendered so. The fly ingests solid materials by mixing a little bit of regurgitated food, which it stores in its welldeveloped ventral crop, with the solid food until desired consistency is reached. The fly then sucks up the solid dissolved in vomitus. Unfortunately, the vomitus is often not entirely sucked up; a small portion is almost always left behind.

This vomitus and the fly's fecal matter are the major sources of micro-organismic infection, although the hairs of its body and the sticky tenent hairs of its feet, which permit the fly to cling to walls upside down, do play a minor role. Ingested infectious organisms can remain viable in the fly's alimentary canal for greater lengths of time and in greater numbers because the digestive tract can provide optimum conditions for bacterial growth. Thus it is obvious that these pathogenic carriers must be checked if disease is to be minimized.

Problems of Present Control Techniques

The ill effects of the pathogenic eating and living habits of the fly demand that the fly's population dynamics be controlled. The two methods of approach to control are killing the existing fly and/or preventing the procreation of future flies by disrupting normal breeding processes. The second approach is obvious; simply take sanitary precautions and remove or reduce the numbers of optimum breeding spots: human feces, animal manure, and improper disposed garbage.

But total eradication through sanitary precautions can never be complete because of the difficulty of achieving totally sterile conditions. More importantly, the time involved between the employment of preventive techniques and the subsequent desired reduction of flies is too long. Therefore, more direct action must be taken via insecticides.

Man's efforts to control the fly and other harmful insects, however, are encumbered by an awkward dilemma. Insects naturally must be controlled but there is rising protest that pesticides are destroying harmless wildlife and endangering even man's life. The toxicity of these pesticides is a result of their fat-soluble and only slowly biodegradable nature. Often organic molecules that are fat-soluble tend to be accumulated in living systems. If not biodegradable, they may be concentrated by the food chain or other mechanisms so that their level in tissue comes to exceed that in the environment by many orders of magnitude. Examples of fat-soluble, slowly degradable compounds are DDT and its related chlorinated aromatic hydrocarbons. Accumulations of these compounds result in a variety of pathological phenomena, including neurological disturbances and birth defects.

However, without control by insecticides, the world might suffer from more alarming consequences. According to the World Health Organization, insect pests are responsible for half of all human deaths due to disease. Insects consume or destroy about a third of everything that man grows or stores. Without pesticides, the U. S. could not maintain its present standard of living.

Unfortunately, pesticides also frequently generate increasing immunity in their intended victims. To the problem of immunity, food-raisers are responding both with heavier applications and with pleas for more potent new chemicals. At the same time, health officials are becoming more sensitive to the toxic dangers of pesticides and their residues and more aware of the concentrations of these chemicals in our food and water. Thus chemists and economic entomologists find themselves in an embarrassing crossfire: on one side, from the farmers demanding more potent insecticides, and on the other, from the health agencies demanding less toxic chemicals.

A New Approach to Control: Pheromones

Public consciousness of the undesirable effects of pesticides, coupled with the pressing problem of harmful insect species becoming resistant and immune to many insecticides, has spurred research into new areas and methods of pest control. The area with most promise, and the one in which I am actively doing research under the directorship of Prof. G. W. Gribble is the use of pheromones to lure insects to lethal traps.

Pheromones are external secretions used by almost every animal, including man, to communicate among their own species. These chemical secretions when either tasted or smelled can bring about prompt behavioral change of the fellow members of the same species. The word pheromone was coined by Karlson and Butenandt from the Greek pherein (to carry) and horman (to excite, stimulate). In earlier literature on the subject, these chemicals were referred to as ectohormones, but unlike true hormones, which are secreted internally to regulate the organism's internal environment, pheromones are secreted externally to regulate the organism's external environment.

The occurrence of this chemical communication system is widespread, Almost every form of insect activity, including congregating, food scavenging, defending, copulating and even the forming of their social castes, is controlled either directly or indirectly by these chemical secretions. Even mammals utilize chemical communication. Every time your dog urinates on a tree or fire hydrant, he is leaving a pheromone that marks out his territory against any intruding dogs. The sex pheromone of our primate cousin, the Rhesus monkey, has recently been isolated from the female's vagina. Even when the female is absent, this pheromone, labeled copulin, will sexually excite the male.

The notion of a chemically based communication system may seem strange to us since human interactions are so strongly based on auditory and visual conventions. Nevertheless, on closer inspection we find that man too, albeit to a lesser degree, possesses a distinct chemical communical system. Apocrine glands located in the armpits and around the genitals secrete oils with musk-like smells (thought to be important in sexual behavior). More than likely, however, man's pheromone exocrine glands have atrophied since he has developed language, a much more versatile mode of communication.

It is our intention to use the insect's very own potent sex pheromone to lure the pests to baited traps for collection or extermination. Since pheromones will attract and excite only insects of the same species, the traps baited with the pheromone lure will be very specific for the pest in question. Instead of spraying the whole countryside with insectide, one could specifically attract the unwanted insect to traps where it would make contact with an appropriate insecticide, sterilizing chemical, or other exterminating device. These traps could also be used for instantaneous detecting of infestations and for determining the size of the pest's population. The odorous lures and the lethal chemicals could then be applied only at the most effective time and place to eliminate any chance of the return of the pest.

One must also consider the desirable side effect of permeating the air with this insect aphrodisiac. If many traps are employed, there would be so much pheromone in the air that the male would be incapable of orienting to the pheromone normally released by the living female. The male, therefore, could never find the female to inseminate. In short, with pheromones, the battle against harmful insects would be much less costly, more efficient, and the problems of contamination of the environment by toxic materials would be vastly reduced.

Already this method has saved the government millions of dollars by helping to eradicate the Mediterranean fruit fly from over a million infested acres in Florida. The use of gyplure, the sex attractant of the gypsy moth, has helped to prevent the moth from spreading outside of the New England area. As more pheromones are isolated and synthesized, hopefully more stories of success will be told.

Research at Dartmouth on the House Fly

A group headed by Morton Beroza of the U. S. Department of Agriculture very recently isolated the sex attractant of the female housefly. The fly was expected to have a pheromone because live and dead female flies were found to be attractive to males. Other studies revealed that male flies were also attracted to the feces of the sexually mature females and to lipid extracts of fly feces and cuticular lipids.

The pheromone was isolated by surface washings of hundreds of flys with hexane and ether. The concentrate was chromatographed and the active component was identified as a long 23 carbon hydrocarbon chain with a cis double bond between the ninth and tenth carbons. The name muscalure was chosen for this pheromone molecule whose technical name is cis-9-tricosene. In limited field trials, twice as many flies landed on a grid treated with 0.0001 grams of muscalure as on an untreated grid. Though not a potent attractant compared to some sex pheromones, muscalure is expected to be inexpensive to manufacture; and it has good potential for reducing the amount of insecticide needed to control the übiquitous housefly.

Dr. Gribble and I have succeeded in coming up with a new commercial synthetic route to muscalure and we are now trying to improve the potency of the fly's own sex lure by slightly modifying the molecule.

The Wittig synthesis1 originally worked out was not practical for commercial use. Not only were the reactants very expensive, but too much time and labor was involved. We also observed the trans isomer which would subtract from the total synthesized amount of the active cis isomer.

Our new synthesis gives only the desired cis isomer. The reactants of this new synthesis—a Kolbe electrolysis2-are cheap fatty acids obtained by treating animal fat with lye in a simple hydrolytic process called saponification: the same process used in making soap. The two fatty acids, oleic acid which naturally has a cis double bond at the nine position, and heptanoic acid are added together on the anode of an electrolytic apparatus. The yields of reaction have been very good and any other by-products are efficiently removed by chromatography.

But we cannot stop research here with just the synthesis of the natural pheromone, we must now look for more potent analogs of the natural lure. There are two main approaches by which we might improve the effectiveness of the lure. First, we can change the molecule slightly to increase the volatility of the lure while retaining its activity. The more volatile compound would then be more competitive for the male's attention than would the female's natural lure. The male would therefore not find the female to inseminate but rather would find a trap.

The second approach to a more effective lure would be slightly changing the molecule in order to block the male's olfactory senses from perceiving the female's lure. Lynn Riddiford at Harvard has evidence that the pheromone is perceived when it binds with a protein in the sensilla trichodea, the olfactory receptors in the antennae. This tightly bound pheromone-receptor protein complex then perhaps initiates dendrite firing, possibly through a mechanism involving disorientation and depolarization of the receptor membrane. If proteins are indeed used to perceive pheromones, then one must assume that the turnover rate of the receptor proteins is very fast. We, therefore, hope to synthesize a pheromone analog that will bind irreversibly with the receptor proteins. In this way, we hope to make the male flies impotent by blocking their receptor proteins from detecting any of the female's pheromone.

Even if we are successful in our pursuit of a better sex attractant than the female's own natural lure, we are still far from winning the battle against the household fly. The entomologists of USDA, with whom we are working, must take over and design the proper trap. In order to get maximum catches, the entomologists must choose the best color, size, type of bait dispenser, proper amount of bait, and trapping technique (which may utilize a volatile insecticide, a sticky substance, a mechanical baffle, or a detergent liquid in the trap).

There are other subtleties too. An attractant found effective in a laboratory bioassay sometimes does not attract in the field. Many times the attractants are masked by other vapors in the air. In addition, we often are encountering the need to use combinations of chemicals so that the proper vapor pressure is present. Ultimately, any attractant that is present must prove itself in the field in direct competition with the many natural odors, colors, and other stimuli present under actual conditions of use. Fortunately, although it is too early to tell, no masking (reduction of activity) by other odors has been noted for muscalure.

Obviously, much more research must be done before we can say we have the upper hand on the insect's control. Perhaps we can come up with a more potent lure, perhaps not. At least with the Kolbe synthesis we have a cheap access to the fly's sex pheromone which will hopefully help control this insect pest and thereby reduce the disease and pestilence that it carries.

APOSTOLI II by Gordon Wallace '74