12 Arthropod Semiochemicals as Multifunctional Natural Products Murray S. Blum CONTENTS 12.1 Introduction 12.2 Multifunctional Semiochemicals 12.2.1 Cantharidin 12.2.2 Multifunctional Queen Pheromones 12.2.3 Venomous Alkaloids 12.2.4 2-Alkyl-6-Methylpiperidines 12.2.5 Pheromones as Interspecific Inhibitors References 12.1 Introduction The number of arthropod species (insects, millipedes, spiders, mites, crabs, and related groups) is truly fulsome, conservatively numbering between 1,000,000 and 5,000,000 spe- cies, and constituting about 80% of all kinds of animals. 1 The beetles alone (~500,000 spe- cies) dominate the insects 1 and the vertebrates as well; there are at least 10 times more beetle species than vertebrate species, 2 and new species of beetles are being described daily. Significantly, a large variety of unique compounds has already been identified as arthro- pod natural products, 3 notwithstanding the fact that relatively few species have been sub- ject to analytical scrutiny. Clearly, for arthropod natural products the best is yet to come and considering what has already been established, the future would appear to be remarkably bright both in terms of structural chemistry and functionality. In this brief review, the ability of arthropods to biosynthesize a variety of novel com- pounds and to utilize these glandular products multifunctionally will be emphasized as critical developments that have enhanced the competitiveness of the arthropods. Particular emphasis will be placed on semiochemicals, signaling agents utilized in intra- and inter- specific contexts. © 1999 by CRC Press LLC 12.2 Multifunctional Semiochemicals Arthropods are the paramount producers of semiochemicals in the animal kingdom and these compounds have been adapted to subserve a variety of important functions for selected groups of these invertebrates. For example, the virtuosity of arthropods as produc- ers of alkaloids chiefly reflects the biosynthetic prowess of ants which generate most of these compounds as poison gland products that often possess diverse functions 4 both inside and outside of the colonial milieu. In short, the success of many arthropod species is clearly identified with both the synthesis and adaptive utilization of semiochemical natural products. In the discussion that follows, representative semiochemicals, each of which has been demonstrated to possess a considerable diversity of important biological roles, will be examined as examples of what has been referred to as pheromonal parsimony. 5 12.2.1 Cantharidin Cantharidin, the anhydride of cantharidic acid (I), is produced by bee- tles in the family Meloidae and a few related families and has been referred to as Spanish fly for hundreds of years. 6 These insects are sometimes described as blister beetles because, when disturbed, they discharge cantharidin-fortified blood from their legs and this exudate can cause severe dermal lesions in vertebrates and also can repel invertebrates. 7 As we shall see later, the powerful vesicatory proper- ties of cantharidin have been indirectly responsible for its widespread use as a putative sexual stimulant. Cantharidin possesses potent antifungal activities; the female beetle is reported to coat her eggs with this compound, a potent growth inhibitor for Microsporum and Trichophyton species. 8 Since the eggs of the beetles are incubated in warm and moist environments which favor the growth of invasive fungi, the main function of this terpenoid anhydride is probably to protect developing blister beetle embryos from insect-attacking fungi. 9 Consistent with this suggestion is the fact that during copulation male beetles actu- ally transfer large amounts of cantharidin to the females as a copulatory “bonus.” 10 The transferred cantharidin is used to coat the eggs in what is almost certainly an antifungal strategy. In addition to its function as both a vesicant and a fungicide, cantharidin has been tested as a powerful anthropogenic agent in a wide variety of human systems, dedicated to either treating pathological conditions or attempting to enhance sexual ambitions. Complete remission of epidermal cancer in pigs has been effected by topical treatment with cantharidin. 11 In addition, this vesicatory anhydride when applied in low doses, resulted in the total remission of dermal cancer in human beings. 12 Cantharidin, which is structurally similar to a well-known herbicide, is highly active as a selective herbicide. 13 Spanish fly has a long history in medicine, having been used by Hippocrates as a cure for dropsy nearly 2000 years ago. 14 This compound was considered to be of great medicinal value and was a major drug in the treatment of bladder and kidney infections and stones, strangury, dropsy, and certain venereal diseases. 15 Painful conditions such as pleurisy and sciatica were routinely treated with cantharidin as further testimony to the high esteem with which this drug was held in medical circles. However, it is the long-standing reputa- © 1999 by CRC Press LLC tion of this compound as a putative aphrodisiac that has led to its use as a sexual stimulant and in some cases, an abortifacient. Although cantharidin in minute doses can cause bladder and kidney irritation and pain- ful voiding of urine with a sensation of burning, these symptoms hardly justify its reputa- tion as a powerful aphrodisiac. Many women have died of cantharidin poisoning. Indeed, the Marquis de Sade was beheaded in absentia for feeding aniseed sweets treated with cantharidin to two prostitutes who identified de Sade before they died. 16 The corrosive action of the anhydride, first seen in skin lesions, is particularly devastating when it is ingested, causing corrosion of tissues in the mouth, especially the palate. Curiously, cantharidin has a completely unexpected effect on the human male. In two instances French troops in North Africa ate the legs of frogs that had eaten copious numbers of blister beetles. The frogs legs contained cantharidin that had been absorbed from the ingested beetles. In both cases the soldiers experienced a potpourri of minor med- ical problems but the troops were particularly inconvenienced by a painful priapism. 17-18 The erections incapacitated the Zouaves as effectively as any enemy action. “The Beetle of Aphrodite” had struck again! 12.2.2 Multifunctional Queen Pheromones Pheromonal parsimony may reach its highest expression in the honey bee, Apis mellifera, in terms of social communication in the arthropods. The mandibular glands of the queen bee biosynthesize a complex mixture of acids and esters which is dominated by a few novel C 10 compounds. The activities of one of these glandular constituents, the queen substance or (E)-9-oxo-2-decenoic acid (9-ODA) (II), appears to be synergized by other compounds pro- duced in the mandibular glands. 19 One of these,(E)-9-hydroxy-2-decenoic acid (9-HDA) (III), also possesses several pheromonal roles of its own, further demonstrating the elegant control of sociality achieved with the queen’s mandibular gland products. The utilization of a limited series of very characteristic natural products as behavioral regulators for both workers and drones has enabled queen bees to “fine tune” — and control — the social structure of populous honey bee colonies. 9-ODA possesses two critical primer activities that are synergized by other compounds such as 9-HDA; neither compound is active alone. Primer pheromones do not produce an immediate response but rather, exhibit a delayed response which may occur after 24 hours or more and frequently involves the reproductive system. For example, ovarian develop- © 1999 by CRC Press LLC ment of the nonreproductive workers is inhibited by 9-ODA, but the workers must have physical contact with the queen in order for the the pheromonal activity to be expressed. 20 Another primer activity results in inhibiting worker activity leading to the construction of queen cells, thus enabling the queen to pheromonally suppress the rearing of potential com- petitors. 21 In addition to these primer functions that occur in the milieu of the hive, remark- able multifunctionality characterizes the roles of 9-ODA (plus synergists) both in and out of the hive. In the hive, workers are attracted to the queen by 9-ODA and form characteristic retinues around her that can result in food and pheromonal exchange. 22 9-ODA also pos- sesses a major releaser role for the workers, but this is expressed outside the hive. The swarming of honey bees resulting in the selection of new nesting sites is controlled by a medley of pheromones. The formation and movement of swarms is regulated by worker (scout) pheromones that are secreted from an abdominal structure, the Nasanov gland. The Nasanov secretion is dominated by a mixture of terpenes (e.g., geraniol, citral, farnesol) that is ideally suited to attract other scout bees to a potential nesting cavity that has been marked with the secretion. 23 Oriented swarm movement is realized if the queen secretes 9-ODA which acts as a short-range attractant for workers. In the absence of the Nasanov terpenes, 9-ODA is unattractive to the swarming bees. 9-ODA has another releaser function, but in this case it is with drone bees rather than with workers. This acid is a powerful sex attractant for male bees, attracting large numbers of drones at altitudes of at least 200 m. 24 Only 9-ODA is required for powerful sex attractancy, in contrast to the need for glandular synergists when this compound functions as a pheromone for workers. 9-ODA is a highly specific sex pheromone, closely related compounds exhibiting no activity whatsoever. 25 12.2.3 Venomous Alkaloids Many groups of ants in the subfamily Myrmicinae characteristically produce poison gland secretions that are dominated by alkaloids, rather than the proteins typical of the ants’ rel- atives, bees and wasps. A large variety of alkaloids are produced by ants and characteristic compounds are identified with different genera. 26 However, while a variety of pharmaco- logically active compounds are biosynthesized by these insects, many are not injected into their assailants. Diverse alkaloid-producing ants do not have a functional sting, and the externalized venom is smeared on adversaries often packing a topical wallop. On the other hand, a variety of ant species attack their enemies by introducing venom by hypodermic injection and the deterrent effects can be considerable. Nevertheless, notwithstanding their method of introduction to an adversary, venomous alkaloids have been demonstrated to possess great multifunctionality as defensive compounds. 12.2.4 2-Alkyl-6-Methylpiperidines Mixtures of cis- and trans-2-alkyl-6-methylpiperidines have been identified as poison gland products of workers and queens of ants in the genus Solenopsis, the trans-isomers generally predominating in the venom of workers. The known dialkylpiperidines in the genus Sole- nopsis possess long n-alkyl groups that are either saturated (IV) or contain a carbon–carbon double bond at the ninth carbon from the terminal methyl group (V). 27 Members of the subgenus Solenopsis are known as fire ants because of the great pain that is associated with the worker sting. A concatenation of toxicological and pharmacological events occurs when Solenopsis venom is introduced subdermally into a human. An exami- nation of the diverse activities of the venom alkaloids emphasizes their multifunctionality as agents of deterrence against vertebrates. © 1999 by CRC Press LLC Stings of Solenopsis workers produce pronounced dermal necrosis followed by the for- mation of pruritic and sterile pustules. 28 The alkaloids liberate histamine from mast cells resulting in considerable algogenicity, a reaction that intensifies the deterrent effective- ness of the alkaloids. 29 In addition to these reactions, the dialkylpiperidines possess powerful lytic activity, instantly hemolyzing mammalian erythrocyes. 30 In addition to these toxicological effects, the alkaloids demonstrate their not inconsiderable multifunc- tionality by exhibiting a great range of pharmacological activities that are unleased against a variety of biochemical systems. These compounds are strong inhibitors of ATPases 31 and in addition, they reduce mitochondrial respiration and uncouple oxida- tive phosphorylation. 32 The ability of the dialkylpiperidines to also block neuromuscular junctions 33 further identifies these compounds as very versatile defensive agents. However, in addition to their activities against pharmacological targets, these alkaloids exhibit considerable semi- ochemical parsimony in a variety of ecological contexts. The poison gland products possess a wide range of antimicrobial properties and, in addition, they are phytotoxic as well, fur- ther identifying these compounds as versatile agents of semiochemical multifunctional- ity. 34 The 2,6-dialkylpiperidines possess powerful antibacterial activity against a variety of species 35 and in addition, these compounds are potent growth inhibitors of diverse fungal species. 36 These alkaloids also exhibit considerable activity as insecticides which compares to that of commercial insecticides. 37 Furthermore, the ability of these compounds to exhibit insecticidal activity when applied either topically or by injection, enables fire ants to be both effective aggressors and predators. The fire ant’s offensive arsenal is further expanded by the well-developed repellency to ants exhibited by the 2,6-dialkylpiperidines, 38 which should allow these ants to suc- cessfully compete with other ants for critical resources. In some cases repellency of dif- ferent ant species is achieved by dispersing venom through the air (gaster flagging), 39 but in the milieu of the nest dispersion of venom by workers is utilized to treat vulnerable fire ant larvae with the antibiotic alkaloids. 40 Similarly, the queen fire ant treats her freshly laid eggs with her poison gland contents, and the concentration of alkaloids is high enough to inhibit the growth of entomopathogenic fungi. 41 The chemical ecology of the fire ants vis-à-vis the 2,6-dialkylpiperidines constitutes a highly adaptive system for both exploiting the acquisition of food and protecting the immatures from intrusive microorganisms. © 1999 by CRC Press LLC 12.2.5 Pheromones as Interspecific Inhibitors Many species of bark beetles in the family Scolytidae have evolved a unique strategy for attacking coniferous trees and utilizing these often formidable plants as sites for mating, feeding, and reproduction. Optimal utilization of the tree as a resource requires that the bark beetles develop a large enough population to overcome the mostly chemical defenses of the target tree. In addition, maximum utilization of the trees’ not inconsiderable resources also requires the establishment of large numbers of immature and adult beetles to convert the tree into an attractive target for additional bark beetles of the same species. This scenario presup- poses that successful colonization of a pine tree will be achieved by a single species of bark beetle. Since bark beetles utilize aggregation pheromones to attract members of their own species, these volatile information-bearing compounds would seem to constitute ideal agents for guaranteeing the specific integrity of the “chosen” tree. And indeed it has been estab- lished that the pheromonal attractants of selected species of bark beetles possess critical mul- tifunctional value as inhibitors of the attraction of competitive beetle species. 42 Aggregative pheromones are frequently synthesized by bark beetles from monoterpenes derived from the host’s oleoresin. Novel mixtures of oxygenated monoterpenes have been identified as aggregative pheromones in species in the genera Ips and Dendroctonus and these compounds have been demonstrated to function, under field conditions, as both intra- and interspecific agents of mutual interruption. 42 For example, colonizing beetles of the species Ips paraconfusus produce an aggregation pheromone that is dominated by the monoterpenes ipsenol (VI), ipsdienol (VII), and cis-verbenol (VIII). 43 These compounds, which are mainly produced by male beetles, constitute a true synergistic pheromone, all three compounds being required for attraction to the host, ponderosa pine. On the other hand, the attraction of another Ips species, I. latidens, which also attacks ponderosa pine, is inhibited by the ternary mixture employed by I. paraconfusus as an aggregation pheromone. 44 Nevertheless, I. latidens responds to both ipsenol alone and to ipsenol plus cis-verbenol. However, when the complete I. paraconfusus aggregation pheromone containing ipsdienol, ipsenol, and cis-verbenol is pre- sented to Ips latidens, the attraction of adults of I. latidens is completely inhibited. Whereas adults of I. paraconfusus convert myrcene to ipsdienol, 45 an important aggregation phero- mone for this species, 43 adults of I. latidens do not produce this oxygenated monoterpene and it functions as a powerful inhibitor of aggregation when they encounter it. Mutual interruption of attraction can occur when species in the same genus inhabit the same host tree. For example, males of I. pini and I. paraconfusus, boring side by side in logs of ponderosa pine, attract fewer beetles of both species than logs containing males of a single species. Furthermore, the response of I. pini is reduced in the presence of either (–)-ipsenol or (+)-ipsdienol, 46,47 the latter compound being the enantiomer of (–)-ipsdienol, the aggregation pheromone of I. pini. Since both ipsdienol and ipsenol are part of the aggregation pheromone of I. paraconfusus, 43 it is probable that these terpenes are primarily responsible for inhibiting the attraction of I. pini. Mutual interruption also can occur between species in different genera that inhabit the same tree. The western pine beetle, Dendroctonus brevicomis, competes with Ips paraconfusus for ponderosa pine, and these species frequently inhabit the same host tree. Verbenone (IX),one of the aggregation pheromones produced by males and females of D. brevicomis, effectively interrupts the attraction of I. paraconfusus to host trees. 48 However, mutual inter- ruption between these species also has been observed, 49 and it would appear that individuals of both species generate cross-specific attractant inhibitors that probably ensure that a newly invaded host tree will not be catastrophically overwhelmed by beetles of either species. © 1999 by CRC Press LLC References 1. Campbell, N.A., Biology, 4th ed., Benjamin/Cummings, Menlo Park, CA, 1996. 2. Koomen, P., van Nieukerken, E.J., and Krikken, J., in Zoologische Diversiteit in Nederland, van Nieukerken, E.J. and Krikken, J. Eds., Biodiversteit in Nederland, Nationaal Natuurhis- torisch Museum, Leiden (in press). 3. Blum, M.S., Chemical Defenses of Arthropods, Academic Press, New York, 1981. 4. Blum, M.S., in Chemistry and Toxicology of Diverse Classes of Alkaloids, Blum, M.S., Ed., Alaken, Inc., Fort Collins, CO, 145-184, 1996. 5. Blum, M.S., in Annu. Rev. Entomol., vol. 41, Mittler, T.E., Radovsky, F.J., and Resh, V.H., Eds., Ann. Rev. Inc., Palo Alto, CA, 353-374, 1996. 6. Howell, M. and Ford, P., The Beetle of Aphrodite and Other Medical Mysteries, Random House, New York, 1985. 7. Cuénot, L., C.R. Seances Soc. Biol. Ses Fil., 122, 875, 1894. 8. Pinetti, P. and Biggio, P., Boll. Soc. Ital. Biol. Sper., 44, 677, 1968. 9. Selander, R.B., Personal communication, 1976. 10. Sierra, J.R., Woggon, W-D., and Schmid, H., Experientia, 32, 142, 1975. 11. Dubois, R. and Ball, M.V., Bull. Acad. Med., 110, 791, 1933. 12. Dubois, R., C.R. Soc. Biol. Ser. II, 97, 48, 1927. 13. Cutler, H.G., Plant Cell Physiol., 16, 181, 1975. 14. Sollman, T., in A Manual of Pharmacology, W. B. Saunders, Philadelphia, 137, 1949. 15. Groeneveld, J., De Tuto Cantharidum in Medicina Usu Interno, Typis J.H. prostant venales apud Johannem Taylor, London, 1698. 16. Dulauré, J.A., Collection de liste des ci-devant Ducs, Marquis, Contes, Barons, etc., Second Year of Liberty, Pamphlet in the collection of the Musée National, Paris, 1794. 17. Vézien, M., Rec. Mém. Med. Chirurgie Pharm. Mil., 4, 457, 1861. 18. Meynier, J., Arch. Med. Pharm. Mil., 22, 53, 1893. 19. Butler, C.G., Callow, R.K., and Johnston, N.C., Proc. R. Entomol. Soc., 155, 417, 1961. 20. Butler, C.G. and R.K. Callow, Proc. R. Entomol. Soc. Ser. A, 43, 62, 1968. 21. Pain, J., Barbier, M., Bogdanovsky, D., and Lederer, E., Comp. Biochem. Physiol., 6, 233, 1962. 22. Slessor, K.N., Kaminiski, L-A., King, G.G.S., Borden, J.H., and Winston, M.L., Nature, 332, 354, 1988. 23. Schmidt, J.O., Slessor, K.N., and Winston, M.S., Naturwissenschaften, 80, 573, 1993. 24. Gary, N.E., Science, 136, 773, 1962. 25. Blum, M.S., Boch, R., Doolittle, R.E., Tribble, M.T., and Traynham, J.G., J. Insect Physiol., 17, 349, 1971. © 1999 by CRC Press LLC 26. Jones, T.H. and Blum, M.S., in Alkaloids: Chemical and Biological Perspectives, vol. 1, Pelletier, S.W., Ed., John Wiley & Sons, New York, 33, 1983. 27. MacConnell, J.G., Blum, M.S., and Fales, H.M., Tetrahedron, 26, 1129, 1971. 28. Caro, M.R., Derbes, V.J., and Jung, R., Am. Med. Assoc. Arch. Dermatol., 75, 475, 1957. 29. Read G.W., Lind, N.K., and Oda, C.S., Toxicon, 16, 361, 1974. 30. Adrouny, G.A., Derbes, V.J., and Jung, R.C., Science, 130, 449, 1959. 31. Koch, R.B., Dessaiah, D., and Ahmed, K., Biochem. Pharmacol., 26, 983, 1977. 32. Cheng, E.Y., Cutkomp, L.K., and Koch, R.B., Biochem. Pharmacol., 26, 1179, 1977. 33. Yeh, J.Z., Narahashi, T., and Almon, R.R., J. Pharmacol. Exp. Ther., 194, 373, 1975. 34. Blum, M.S., Walker, J.R., Callahan, P.S., and Novak, A.F., Science, 128, 306, 1958. 35. Jouvanez, D.P., Blum, M.S., and MacConnell, J.G., Antimicrob. Agents Chemother., 2, 291, 1972. 36. Cole, L.K., Antifungal, insecticidal, and potential therapeutic properties of ant venom alkaloids and ant alarm pheromones, Ph.D. thesis, University of Georgia, 155, 1974. 37. Escoubas, P., Alcalöides de fourmis: identification, toxicité et mode d’action, Ph.D. thesis, University Pierre et Marie Curie, 145, 1988. 38. Blum, M.S., Everett, D.M., Jones, T.H., and Fales, H.M., in Naturally Occurring Pest Bioregulators, Hedin, P.A., Ed., ACS Symp. Ser. 449, Washington, D.C., American Chemical Society, 14, 1991. 39. Obin, M.S. and Vander Meer, R.K., J. Chem. Ecol., 11, 1757, 1985. 40. Blum, M.S., in Bioregulators for Pest Control, Hedin, P.A., Ed., ACS Symp. Ser. 276, Washington, D.C., American Chemical Society, 393, 1985. 41. Vander Meer, R.K. and Morel, L., Naturwissenschaften, 81, 682, 1994. 42. Wood, D.L., in Annu. Rev. Entomol., vol. 27, Mittler, T.E., Radovsky, F.J., and Resh, V.H., Eds., Annu. Rev. Entomol., Palo Alto, CA, 353-374, 1982. 43. Silverstein, R.M., Rodin, J.O., and Wood, D.L., Science, 154, 509, 1966. 44. Wood, D.L., Stark, R.W., Silverstein, R.M., and Rodin, J.O., Nature, 215, 206, 1967. 45. Hendry, L.B., Piatek, B., Browne, L.H., Wood, D.L., Byers, J.A., Fish, R.H., and Hicks, R.S., Nature, 284, 85, 1980. 46. Birch, M.C., Light, D.M., and Mori, K., Nature, 270, 1977. 47. Birch, M.C., Light, D.M., Wood, D.L., Browne, L.E., Silverstein, R.M., Bergot, B.J., Ohloff, G., West, J.R., and Young, J.C., J. Chem. Ecol., 6, 703, 1980. 48. Browne, L.E., Wood, D.L., Bedard, W.D., Silverstein, R.M., and West, J.R., J. Chem. Ecol., 5, 397, 1979. 49. Byers, J.A. and Wood, D.L., J. Chem. Ecol., 6, 149, 1980. © 1999 by CRC Press LLC . substance or (E )-9 -oxo-2-decenoic acid (9-ODA) (II), appears to be synergized by other compounds pro- duced in the mandibular glands. 19 One of these,(E )-9 -hydroxy-2-decenoic acid (9-HDA) (III),. to possess great multifunctionality as defensive compounds. 12. 2.4 2-Alkyl-6-Methylpiperidines Mixtures of cis- and trans-2-alkyl-6-methylpiperidines have been identified as poison gland products. Cantharidin 12. 2.2 Multifunctional Queen Pheromones 12. 2.3 Venomous Alkaloids 12. 2.4 2-Alkyl-6-Methylpiperidines 12. 2.5 Pheromones as Interspecific Inhibitors References 12. 1 Introduction The number of arthropod