California Association of Chemistry Teachers Charles E. Berkoff Smith Kline and French Laboratories
1500 Spring Garden Street Philadelphia, Pennsylvania 19101
I
I
Insect Hormones and Insect Control O r , sex and the single pyrrhocoris apterus
S i r Vincent Wigglesworth (I) who in 1934 established the existence of insect hormones once said (2) "The virtue of the insects as a medium for the study of physiological chemistry lies in their diversity. Given sufficient acquaintance with them, it is generally possible to find material ideally suited to the study of any problem." This may be something of an exaggeration perhaps, but t.here is an element of truth there. Recent advances in the chemistry and biochemistry of insect hormones have been particularly rapid and exciting. Summarizing some highlights of these developments (3) may be more instructive than going into any great chemical depth. I would like to limit this coverage to a consideration of Brain Hormone (BH), Juvenile Hormone (JH), and Molting Hormone (MH), three metamorphosis hormones which play vital roles in the regulation of postembryonic development,. Hormonal control of insect development may be schematically represented by Figure 1. In essence, BH stimulates the secretion of M H while J H is secreted independently. The presence or absence of these three hormones governs the pos&embryonic development of the insect. Brain Hormone (BH)
Molting in insects is initiated by neurosecretory cells of the brain which secrete a prothoracotropic hormone. This is Brain Hormone, and it stimulates the prothoracic gland to release Molting Hormone. It has been suggested that BH has a direct action on tissues, acting synergistically with MH. The early chemical studies on B H for the most part took place in Japan. In 1962, Kobayashi and his coworkers (4) isolated 4 mg of a crystalline substance by painstakingly dissecting nearly 1/4 million "brains" of the silkworm, Rombyx mori, and apparently demonstrated the hormonal activity of the extract. Subsequent studies (6) established unequivocally that the isolated material was-cholesterol. That BH was purportedly cholesterol was in direct conflict with other published views. For example, Ischikawa and Ishiaaki (6, 7) demonstrated that BH was a protein. Furthermore, cholesterol is a normal constituent of the insect diet and would, perhaps, not be expected to exert horThis talk was presented at the California Association of Chemistry Teachers Summer Conference held in Asilomar, California, in August 1970 and was also presented in part at the Intra-Soienoo Research Foundation's Symposium honoring 1970 Medalist Professor Carl Djernssi held in Santa Monica, California, Jsnuary 1970.
monal effects. I t was subsequently demonstrated that pure cholesterol had no effecton molting (8). A third view of the chemical nature of BH has been presented by Gersch (9). From both the central nervous system and the cerebrum of the cockroach, Periplaneta americana, Gersch has isolated the neurohormones CI, Dl, C2,and D1, where only neurohormoue Dl, a crystalline peptide, had demonstrable metamorphosis activity. A popular but unpublished view is that there are possibly two (or even three) Brain Hormones. A number of substances have been claimed to have BH-like activity (10, I f ) , but in the absence of a reliable bioassay serious consideration of these claims is questionable to say the least. Clearly we need a good bioassay for BH-activity before the chemist can address himself to any elaboration of the BH molecule. Juvenile Hormone (JH) Function
Juvenile Hormone is, as shown in Figure 1, secreted by the corpora allata; many functions have been attributed to it. The most striking property of J H is unquestionably its morphogenetic activity. J H prevents the metamorphosis of immature insects by acting in concert with Molting Hormone to maintain the juvenile or larval character of the growing insect. It has also been claimed that the corpus allatum hormone, J H that is, can induce reversal of adult to larval integument (12). This is an apparent reversal of the ageing process smacking a little of science fiction. In Figure BRAIN
BH
\ Corpur Cardiac"
JH
/-->
\ \ \
Figure 1.
Hormonal control of insect development.
Volume 48, Number 9, September 1971
/
577
Figure 2.
Effect of JH applied topically to the pupo of the Cynthia moth.
application of mass spectral analysis which contributed so much to the success of their mork. Following Roller's (17) synthesis of J H a number of more or less sophisticated syntheses have appearedsome deliberately stereospecific, like W. S. Johnson's (19), E. J. Corey's (ZO), and the Syntex group's @I), others deliberately designed to lead to a variety of related structures which could then be tested for hormonomimetic activity. There are probably a s many as 10 synthetic sequences known. I will not elaborate on the detail of these; let me instead say a little about other compounds with JH-activity, since, from the insect control viewpoint, this is really where most of the action is. Historically, the very first compounds with demonstrable (although veak) JH-activity, farnesol (the alcohol) and farnesal (the corresponding aldehyde), were isolated by Schmialelc (22) from the faeces of the beetle, Tenebriomolitor. (R = CH,OH (farnesol); R = CHO (farnesal); R = CO,H (farnesoic acid).)
2 yon see the effect of J H applied topically to the pupa of the Cynthia moth. The result is a pupal-adult intermediate, where one sees a normal adult head and thorax, and a typical pupal abdomen. Chemistry of JH
I n 1956, Carroll Williams (15) of Harvard announced that the abdomen of the male silkmoth, Platysamia cecropia, as an exceptionally rich source of JH. Noting that topical application of J H caused insects to die vithont completing their development, Williams recognized t,he pol~nrfnlinsecticidal potential of JHpowerful, since insect,s could scarcely develop resistance to their o~r.11hormones. Williams, and his chemical colleague J.H. La\\- (14) went on t o purify the original cecyopia extract some 50,000-fold, establishing that the major component of the material mas the 9,lOepoxide of methyl hexadecanoate. They synthesized both cis and trans isomers to see xhich n.as J H , and lo and behold-neither was! They had f o l l o ~ e d the wrong fraction-a case perhaps of being unequal before the La~v. Success was to await the efforts of Roller and his colleagues at Wisconsin. They too purified the cecropia extract lP-fold (12, 16), hut n-ent on to achieve characterization and synthesis of the J H molecule (16, 17), methyl trans,trans, cis-l0-epoxy-7-ethyl-3,1l-dimethyl2,6-t,ridecadienoate (I)
I
As yo11 see, dH is a relatively simple molecule-not quite a sesquiterpenoid, hut almost. This is the 7ethyl derivative. Kot too long ago the 7-methyl analog was also isolated from the cecropia moth and it, too, has JH-activity (18). I will not expand on t,he details of Roller's very elegant structural &dies, but it should he noted that he and his covorkers achieved their success using microgram quantities of JH. They were, therefore, forced to put to full use all modern physico-chemical techniques which are virtunlly essential for the solution of such problems. Above all, I think, it was their telling 578
/
lournol of Chemical Education
Evaluat,ion of the .TH-activity of a wide variety of farnesol derivatives, sesquiterpenoids in general, and both natural and synbhetic compounds followed (25, 24). In 1966, Law, Yuan, and Williams (25) described the synthesis of a neutral product of very high JH-activhy by treading farnesoic acid with ethanolic HC1. The crude product said to be a mixture of at least six compounds (although I have heard >20 at the last count), blocked the emergence of the adult Yellow Fever mosquito, Aedes aegypti; they used 1 part crude reaction product in 100,000 parts \~ater. Lethal effects were also exerted on the hnman body louse, Pediculus humanus the vector of epidemic typhus, trench fever, and epidemic relapsing fever (26). Using in plac_e of farnesoic acid t,he corresponding methyl ester Sorm and his colleagues (27) at the Czechoslovak Academy of Sciences reported the isolation of an essentially homogeneous product-the dichloroester, 111. I t is described as methyl farnesoate "dihydrochloride" in the literature. Slam& (28), also of the Czech Academy, has now described spectacular results wit,h this dichloro compound which could have profound insecticidal significance. His initial studies showed that as little as 1 pg of t,he dichloroester per specimen is enough to induce permanent sterilit,y in the adult female of Pyrrhocoris aptems, also known as the Linden bug, and a pest by many standards. These studies were extended to the treatment of males of Pyrrhocoris. When mated with est,er-treated males, females laid completely sterile eggs for the remainder of their lives. Now classically, in using the sterilized male technique to control a polygamous insect population, a female is still very likely to meet at least one healthy parher during her repeated matings. SI&ma's technique is such that a t,reated male will render a female sterile no matter how
many normal males she mates with subsequently. Numbered are the days of the winging, swinging Pyrrhocoris apterus. A particularly fascinating chapter of JH-like compounds, again involving Pyrvhoco~isapterus and SlAma, began when the young Czech arrived at Harvard, bug in hand, for a stay with Williams. Together they observed (29) that the Pyvrhocolr's apterus brought from Prague t o Boston failed to undergo normal metamorphosis; without exception all the animals died wit,hout completing their development. Following an impressive piece of detective ~ o r where k a dozen or so possible explanat,ionsfor this observation were considered, evaluated, and rejected, the source of JH-activity was eventually t,raced to exposure of the bugs to a specific brand of paper towel placed in the rearing jars. Following t,he evaluation of the JH-like activit,y of other samples of paper it was solemnly declared t,hat T h e N e w Y o l k Times, The W a l l Street Journal, and Science were active while T h e Times, that's T h e Times (of London), and Natuve r e r e inactive ! It was subsequently discovered that t,he active principle in American paper, the so-called "Paper Factor," originated in the balsam fir, Abies balsamea. Bo~versand his colleagues (30) a t the Department of Agriculture in Reltsville vent on to isolate, purify, and characterize this activc principle, (+)-juvabione, showing it to be the methyl ester of todomatuic acid (IV).
Chemistry o f M H
Our structural knowledge of ecdysone is almost entirely due t o Karlson and his coworkers in Germany. The characterization of M H was known to be a gargantuan task at the outset, and in 1954 Jcarlson and Butenandt (97) reported the isolation of 25 mg (heaps of material by today's standards) of the crystalline hormone, ecdysone, from 500 kg of the pupae of the commercially used silkworm, Bombyx mori. Eleven years later ecdysone was declared to be the pentahydroxycholestenone (VI) (28, 38, 14a, 22R, 25-pentahydroxy-A'-58-cholesten-6-one) (58)
VI
Confirmation of the structure by unambiguous, straight-forward synthesis was subsequently announced almost simultaneously by two groups, Syntex (SO), and the combined forces of Schering (Berlin) and HofmannLaRoche (Basel) (40). Such has been the fierce spirit of competition in this whole area, that the ScheringRoche group won by just 3 days (41). MH-Like Substances
The dehydro derivative, (V), is additionally present in balsam fir indigenous to Czechoslovakia, and it t,oo shorn JH-activity (51). Molting Hormone (MH) Function
We now return to Figure 1 to consider the third of the three met,amorphosis hormones I initially mentioned, Molting Hormone, or, as it is called in its pure crystalline state, ecdysone. It is produced in the prothoracic (or ecdysial) gland by st,imulation of BH. Transformation to the pupa occurs when J H levels are lour enough and MH is secreted alone. Imaginal development is controlled by MH, possibly in synergy n+h R -H - ...-. ~ A number of different actions have been attributed to RIH, perhaps the most dramatic being its ability t o induce protein synthesis (52). At December's meeting of the Entomological Society held in Chicago Gilbert (55) presented evidence for the R4H-actiGation of a gene to produce messenger RNA. Clever (54) describes the primary steps in the cellular response to ecdysone as Eedysone + (?) -+ Activation of -+ m-ltNA Specific Genes I
Further Gene Activation
= (?) -ProteinJ.
I t is somewhat controversial ivhet,her ecdysone acts directly or indirectly on the gene (55,56).
A number of naturally occurring compounds that mimic the activity of R'IH have now been isolated and characterized. To date, these compounds are all steroids structurally related to the ecdysone molecule. For example, from one ton of the crayfish, Jasus lalandei, Horn (42) in Australia isolated 2 mg of pure crustecdysone. We now know that crustecdysone, VII, has a number of synonyms depending on its source: crustecdysone (42); 8-ecdysone (45); ecdysterone (44); 20-hydroxyecdysone (44); polypodine A (45); isoinokosterone (46).
HO
W
One of the sources of this simple monohydroxylated derivative of ecdysone is Bombyx mori pupae; that is, it co-exists nith ecdysone (45). But a new dimension in natural product chemistry mas created by Nakanishi's discovery of MH-like substances not in insects or in crustacea, but in plants (47). Podocarpus elatus, an Australian timber tree, is, for example, a "rich source" of crustecdysone (48), and many other MHactive phytosteroids have been and continue to be isolated and characterized (5). Most of the 35 or so MH-active plant steroids now known are described by structure VIII. Volume 48, Number 9, September 1971
/
579
VIII
(R,R1, and R": various combinations of H, alkyl, and hydroxyalkyl.) Role of Plant JH- and MH-active Substances
The question of why plants synthesize JH-active compounds like juvabione and dehydrojuvabione, and MH-active compounds like the steroids shown has received comment,. Williams and S l h a (29) expressed the view that the balsam fir, which is known to be resistant to P y ~ r h o c o ~aptems, is had evolved an extremely sophisticated defensive system against insect predators sharing the endocrine sensitivities of P. aptems. Galbraith and Horn (48) have suggested that Podocarpus elatus, also known to be particularly resistant to insect attack, elaborates ME-active steroids to int,erfere with the grorth processes of insect predators. It seems to me that a simpler and in many ways more att,ractive explanation for the presence of JHand MH-active compounds in plants could rest on t,heir st,ructuralsimilarity to plant sex hormones. Two plant sex hormones, sirenin, IX, (49) and antheridiol, X, (50),have recently been isolated and characterized.
Now let us compare structurally dehydrojuvabione, XI, (or, of course, juvabione) with sirenin, IX.
By writing dehydrojuvabione this way, I think you can see that t,he same basic C-skeleton is common to both structures-it is just the oxidation levels that are different. As is seen in X antheridiol is a steroid, the first synthesis of which was recently described (51). This hormone may be structurally compared to a number of MH-active phytosteroids, especially, perhaps, those like cyasterone, XII, (5g) and capitasterone XIII, (53) which, like antheriodiol, X, bear lactone rings in the side chain. 580
/
Journal of Chemical Education
From these comparisons two points emerge. Firstly, an examinat,ion of possible reciprocal biological act,ivities might prove to be instruct,ive. Secondly, t,he plant need not elaborate molecules of t , h juvabione ~ and MHlike steroid types for defensive purposes; it could be that these or related compounds have a role to play in the plant's reproductive processes, and that their defensive properties, if any, are coincidental or perhaps secondarily contrived. Insect Control
We have considered, at least superficially, what these three hormones are, v h a t they do, and why nature apparently produces mimics of them in plants. Whether one is an industrial scientist or not an appropriate question is bow can one apply this information. What does one do with it apart from having it generate more of the same? Although much of our knowledge of insect hormone chemistry and biochemistry can be effectively translated to mammalian physiology and to other areas concerned with natural product chemistry, it is surely to insect control that we must first turn. The challenge of developing selective, effective, and safe insect control is one of the most pressing environmental problems before us today. As you all know Carsonian concern over the harmful effects of DDT residues now confronts most nations in the world. I believe that that concern will markedly increase. Pollution will be or i s surely high on that list where industrial waste, vehicular exhaust, and presently available insecticides will be and should be obvious candidates for attack. Before we do attack pesticides across the board, h o w ever, let us realize that even with the over-kill usage of currently available insecticides, annual damage in the U.S. attributable to insects is some $4 billion. Worldwid6 the figure is in excess of $30 billion. But it is not only for the agricultural industry's sake that we use insecticides [although they'll admit to sales of $1.25 billion in 1968 with a predicted growth potential of $5 billion by 1980 ( 6 4 ) ) We have disease-carrying insects to fight: the vectors of malaria, typhus and many viral diseases. Furthermore, in the absence of insecticides, production of livestock would quickly drop 25%; production of food crops would decrea3e by 30'%; and food prices mould increase by 50-75%. A lot more people would starve to death-and that assumes we stop this senseless, immoral breeding of more
and more people. I guess if we cantinue to peoplepollute, the presence of absence of other pollutants is somewhat academic anyway. But let us look on the bright side, and assume we can learn to control ourselves. If this is so, then we will need, with increasing urgency, new approaches to the problem of insect control. I am convinced that one new approach will come about from our understanding of and ability to interfere with the hormonally mediated insect growth and differentiation processes. This of course will need to be done on a highly selective basis. Of the some 3 million species of insects known only O.lyoare agricultural pests or vectors of human and/or animal disease (65). I n the U.S. there are some GOO undesirable species; many of the remainder are necessary for the preservation of a reasonable level of ecological balance. It is a sobering thought to realize that the number of insects alive is about a billion billion-that is 1018. One out of every three animals in the world is a beetle. Based on his studies with J H , Williams (IS) proposed, as long ago as 1956, the use of hormone-like substances for the control of insect pests, later referring (55) to them as "third generation insecticides" (the first generation is exemplified by lead arsenate; the second by DDT). Since then J H and JH-active compounds have been the focal point for this approach. Ayerst Laboratories in Montreal announced just a few months ago that they can make J H (albeit isomerically impure) in kilogram quantities (56). Thus bulk supplies of a t least 10% active J H will now seemingly be available for broad biological evaluation. It seems to me that an alternate approach could rest on our understanding of insect hormone antagonists. Consider again the sequence of events shown in Figure 1.
If instead of maintaining J H levels we could block MH, then we could again prevent the development of the adult insect. The only known M H antagonists are synthetic steroids (57), typically 3-hydroxy-6-keto derivatives (58) and at this point it is hard to see their general application as insecticides. Even more appealing than antagonizing M H is preventing its secretion; that is, preventing BH from stimulating the production of M H from the prothoracic gland. According t o Gersch (a), there are naturally occurring BH-antagonists (at least in Periplaneta americana). I t would therefore seem reasonable t o test potential BH antagonists for effects on insect growth and development. Finally, let me draw your attention t o anot,her antagonist that could also be very relevant to insect control. According to Bowers (59) there is in normally diapausing beetles a humoral J H antagonist. Although JH-active substances (60)canterminate diapause, it may he that structural knowledge of the J H antagonist could lead us into new areas of challenging chemistry and biochemistry. Literature Cifed ( 1 ) W l a o m s w o n ~V ~ . B.. Qua7t. J . Miwosc. Sci.. 7 7 , 1 9 1 (1934). V . , in "Aspeots of Inseot Biochemistry." Bioehemioal ( 2 ) W~GGLESWO~TW. Sooiety Symposium No. 25, (Editor: GOODWIN. T . W.) Aoademio Press. London, 1965, D xi.
..,... ~ ....,. (28) M m s ~ n P.. , S ~ M *I
