The chemistry of antipredator defense by secondary compounds in neotropical lepidoptera: facts, perspectives and caveats

Journal of the Brazilian Chemical Society

Universidade Estadual de Campinas, Campinas, Brazil¹  Trigo, José R.¹ 

关键词: pyrrolizidine alkaloids;    tropane alkaloids;    aristolochic acids;    cardenolides;    cyanogenic glycosides;    glucosinolates;   

DOI  :  10.1590/S0103-50532000000600002

学科分类：化学（综合）

来源: SciELO

【 摘 要 】

Chemical defense against predation in insects, particularly in Lepidoptera, is a well studied subject in chemical ecology with several reviews available1-10. As defined by Brower8, "chemical defense can be suggested when individual prey organisms contain one or more noxious chemical substances which facilitate proximal and/or distal rejectiona by predators; rejection can occur after a predator partially to completely ingests one or more prey individuals, or after the predator simply smells or tastes the prey".The subject of chemical defense involves various areas of biology and chemistry. From a biological perspective, reports of prey rejection by predators have appeared since the nineteenth century. Bates11 and Müller12 were the first authors to propose that brightly colored butterflies were unpalatable to visually oriented predators, and that similarly conspicuous coloration in other palatableb or unpalatablec Lepidoptera evolved in order to enhance their protection through predator learning. Poulton13 pointed out that the unpalatability of butterflies was derived from their larval host plants. In the last 60-80 years chemical defense has been repeatedly tested against both vertebrate and invertebrate predators1,8,14-17. Evolutionary explanations for the reason why insects acquired noxious chemicals from host plants (so-called substances of secondary metabolism) began to take form after the seminal paper of Ehrlich & Raven18, who proposed a theory of "radiation and escape between plants and butterflies"d. In their scenario, three main steps promoted the diversification of both based mainly on evolution of protective chemicals in the plants: 1. plants with random mutations and recombinations could produce several chemical compounds not directly related to their basic metabolic pathways; 2. some of these compounds, by chance, would protect plants against attack by herbivores; the plants would then enter a new adaptive zone, promoting evolutionary radiation; 3. if insects had also random mutations and recombinations that enabled them to explore these new plant groups, selection would carry them into a new adaptive zone, where they would be free from competitors and natural enemies, promoting again an evolutionary radiation.Chemical defense in insects involve several research areas and the investigations generally assume interdisciplinary feature. Exemplifying this multiplicity we can find studies on physiological mechanisms of biosynthesis and sequestration of defensive compounds by Lepidoptera21,22, evolution of warning coloration associated with unpalatability23-26 and techniques for isolation and identification of the defensive chemicals27.The purpose of this review is to examine the progress in studies of secondary compounds thought to be involved in the chemical defense of Neotropical Lepidoptera. I organized it by classes of chemical compounds, focusing on three aspects: 1. field and laboratory observations on rejection of butterflies and moths by predators, 2. correlation between unpalatability and chemicals found in these insects, and 3. bioassays that test the activity of these chemicals against predators. Perspectives and directions for further research on the subject are suggested. Chemical Compounds Acting as Defense in Neotropical LepidopteraMost organisms have alternate metabolic pathways in addition to those of primary metabolism that involve polysaccharides, lipids, proteins and nucleic acids. The natural products coming from such pathways are called "substances of secondary metabolism"28. In plants, from which butterflies and moths often sequester many of these substances, there are three principal building blocks for these compounds: 1. acetate, which via the mevalonate pathway leads to mono-, sesqui-, and diterpenes, iridoid glycosides and cardenolides; 2. amino acids, leading to cyanogenic glycosides, glucosinolates, pyrrolizidine alkaloids, tropane alkaloids and glycosidase inhibitors; and 3. shikimic acid, the precursor of many aromatic compounds such as furanocoumarins, aristolochic acids and b-carboline alkaloids (via aromatic amino acids). These substances take part in the chemical defenses in Lepidoptera and their roles will be discussed in detail in the next sections.Iridoid glycosidesIridoid glycosides29 (Figure 1, 1) are cyclopentenoid-monoterpene derived compounds in which the glycoside often occurs as an O-linked glycoside at C-1. They occur in about 57 plant families, and more than 600 iridoids structures have been described29.  These compounds have only been investigated in North American butterflies and moths. They are sequestered from their host plants by larvae of the nymphalid Euphydryas phaeton (host plants: Chelone glabra, Aureolaria flava ¾ Scrophulariaceae and Plantago lanceolata ¾ Plantaginaceae), E. chalcedona (Scrophularia californica ¾ Scrophulariaceae), E. anicia (Besseya plantaginea, Castilleja integra ¾ Scrophulariaceae), Poladryas arachne (Penstemon virgatus ¾ Scrophulariaceae) and Junonia coenia (Plantago lanceolata), the pterophorid moth Ptatyptila pica (Castilleja sulphurea), the geometrid moth Meris alticola (Besseya plantaginea) and the sphingid moth Ceratomia catalpae (Catalpa bignonioides)10,29,30. Euphydryas and Poladryas retain the iridoids through the adult stages, while in the remaining species these compounds seem to be lost in the pupal stage10,29,30. Both adult and larva are warningly colored in Euphydryas, while in Junonia and Ceratomia larvae are conspicuous but the adults cryptic, suggesting that in the former both stages would be protected against predators, and in the latter only larvae would. Bowers and collaborators31-33 postulated that due to sequestration of iridoid glycosides from host plants the adults of the genus Euphydryas are generally unpalatable to birds. Bioassays with ants and spiders also demonstrated the role of iridoid glycosides in the chemical protection of larvae34-37.In Neotropical environments Chai38 verified that adults of Thessalia ezra, a melitaeini butterfly that feed on Acanthaceae, was sight- and taste-rejected by birds, but no iridoid glycoside analyses were done. The investigation of all developmental stages of Thessalia and other butterflies that also feed on Acathaceae (e.g. Siproeta, Ortilia, Eresia and Anameca) and Plantaginaceae (e.g. Junonia) will be necessary to elucidate the role of iridoid glycoside in Neotropical species.CardenolidesCardenolides or cardiac glycosides (Figure 1, 2) are, together with pyrrolizidine alkaloids, one of the best studied chemical defense system in insects, particularly in Lepidoptera39. The biosynthetic pathway of these compounds is not completely understood; cholesterol and b-sistosterol are metabolized in plants to pregnenolone, progesterone, and thence to cardenolides28. These compounds are found in 202 plant species in 55 genera and 12 Angiosperm families39.The sequestration of cardenolides by North American Danaus and the rejection of these butterflies by birds have been studied for more than 40 years since the Browers40,41 showed that birds rejected the monarch butterfly D. plexippus. The presence of cardenolides in butterflies was shown to be highly effective against predation by Blue Jays (Cyanocitta cristata bromia, Corvidae). When fed with adults of D. plexippus reared as larvae upon a cardenolide plant, Asclepias curassavica (Asclepiadaceae), the birds exhibited typical effects of cardenolide poisoning, including repeated vomiting42. Monarchs reared on plants bearing cardenolides were much more emetic (= causing vomiting) than those reared on an asclepiad species lacking cardenolides41.Some questions remain open about this system. For example, studies on the role of cardenolides in chemical protection of larvae against predators have received little attention. The presence of two kinds of chemical defense, cardenolides and pyrrolizidine alkaloids, in Danaus species44,45 is poorly explored from either a mechanistic or an evolutionary point of view. The dynamics of cardenolides in Neotropical species of Danaus need to be studied in relation to those found in the North American species.Cyanogenic glycosidesCyanogenic glycosides46 (Figure 2, 3-7) are O-b-glyco-sides of a-hydroxynitriles (cyanohydrins) biosynthetically derived from amino acids; these compounds have intermediate polarity and are water-soluble. They are accumulated in vacuoles in the plant and maybe to be so in animal cells. They generally co-occur with b-glycosidases and hydroxynitrile lyases, which are compartmentalized in other cells. The enzymatic cleavage of cyanogenic glycosides releases HCN plus sugar and ketones or aldehydes. The distribution of these compounds includes at least 2,650 plants (more than 550 genera and 130 families), with Passifloraceae as one of the main families. These compounds are also found in butterflies belonging to the Neotropical genera Heliconius (Nymphalidae, Heliconiinae), and Actinote, Altinote and Abananote (Nymphalidae, Acraeinae) 47,48.  Heliconius uses Passiflora species (Passifloraceae) as larval food plants47, and both larvae and adults biosynthesize de novo, from the amino acids valine and isoleucine, simple cyanogenic glycosides (linamarin and lotaustralin, 3 and 4, respectively ¾ Figure 2)49. Passiflora species have a vast array of different cyanogenic glycosides, varying from simple aliphatic and aromatic compounds to sulphates and cyclopentenoid derivatives46,47 (Figure 2, 6 and 7 respectively). It has recently been demonstrated that a monoglycoside cyclopentenyl cyanogen was sequestered by Heliconius sara fed on Passiflora auriculata50. Moreover, it was found that H. sara has saurauriculatin (8), a thiol derivate from the cyclopentenoid cyanogenic glycoside epivolkenin (7), suggesting that the replacement of the nitrile group by a thiol would prevent cyanide release from the host plant50.Into the neotropical acraeines, Brown and Francini48 showed that 16 species of Actinote, 12 of Altinote and one of Abananote may biosynthesize de novo these compounds in all developmental stages, since their larval host plants (mostly Eupatorium and Mikania, Asteraceae) do not have cyanogenic glycosides.Heliconius species, together with Danaus (Nymphalidae: Danainae), are among the most studied species in relation to unpalatability. Several tests have demonstrated that they are unpalatable to vertebrate predators38,41,51-53. Chai38 verified that Actinote anteas and A. lapihta were sight-rejected by birds. However, there is much speculation in relation to the role of cyanogenic glycosides in chemical defense. The activity of these compounds against predators is poorly understood.GlucosinolatesGlucosinolates (Figure 2, 9) are sulfur- and nitrogen-containing compounds biosynthesized through amino acid metabolism and are found mainly in the order Capparales (e.g. Cruciferae and Capparidaceae)54. Glucosinolates are known for their deterrent activity in plants against generalist herbivores and other natural enemies54. Their volatile derivatives are used as cues by specialist herbivores in the search of host plants and by parasitoids that attack insects feeding on glucosinolate-containing plants54. There are sparse data in the literature showing sequestration of glucosinolates by butterflies or moths from host plants and their role against predators.Many Neotropical pierine butterflies (Appias, Ascia, Leptophobia, Itaballia, Pieriballia, Perrhybris)55,56 use Cruciferae and Capparidaceae as host plants, many of which may contain glucosinolates. Chai38,53 observed that the Neotropical Pierinae Melete, Appias, Perrhybris and Ascia were sight- and/or taste-rejected by birds. In experiments carried out in our laboratory it was verified that larvae of Ascia monuste, which feed on the crucifer Brassica oleracea, were taste-rejected by chicks. In both cases no chemical analyses were carried out to verify if glucosinolates were responsible for this activity.Pyrrolizidine alkaloidsPyrrolizidine alkaloids are probably the best studied defensive compounds in insects, especially in Lepidoptera. Many reviews on the activity of pyrrolizidine alkaloids in chemical defense and the role of these alkaloids in pheromone biosynthesis in Lepidoptera are available21,22,57-62.Pyrrolizidine alkaloids are a diverse class of natural compounds based on a [3.3.0] azabicyclo ring, generally occurring as esters of a "necine base" with "necic acids" as mono- or diesters (Figure 3, 10-13)63. These alkaloids are known mainly from Asteraceae (tribes Eupatorieae and Senecioneae), Boraginaceae, Fabaceae (mainly in Crotalaria), Apocynaceae (subfamily Echitoideae, tribe Parsonsieae) and Orchidaceae (a few genera)21,60,63,65. They are postulated to occur in plants and Lepidoptera as N-oxides21,66, but recent work has discovered more polar metabolites in Ithomiinae butterflies67, similar to glycosylated pyrrolizidine alkaloids that have been characterized in Chrysomelidae bettles68.  Eisner14 was the first to point out the role of pyrrolizidine alkaloids as responsible for chemical defense of the arctiid moth Utetheisa ornatrix against the orb-weaving spider Nephila clavipes. Vasconcellos-Neto and Lewinsohn69 observed that the spider released, unharmed, Ithomiinae and Danainae butterflies from their webs. Brown15-17 found that pyrrolizidine alkaloids acquired from plants visited by adultse were responsible for this activity, since most Ithomiinae and Danainae do not fed as larvae on plants containing pyrrolizidine alkaloid. Other authors have shown the activity of pyrrolizidine alkaloids in other butterflies and moths against spiders74-78, lizards79 and birds79,80. Pure pyrrolizidine alkaloids were bioassayed against spiders81 and birds79,80; N-oxides were shown to be more active than free bases81-83. Corroborating the activity of pyrrolizidine alkaloids against predators, it is known that predators avoid or taste-reject danaine and ithomiine butterflies38,41,53. However, the role of glycosilated alkaloids against predators remains unknown.Aristolochic acidsAristolochic acids (Figure 4, 14) have been found only in plants belonging to the family Aristolochiaceae; biosynthetically, they are nitrophenanthrenes derived from aporphine alkaloids84. The unpalatability of these compounds has been postulated by several authors, but only one bioassay has been done with pure aristolochic acid, where the Japanese tree sparrow Passer montanus rejected rice grains treated with these compounds85,86. However, the authors pointed out that aristolochic acids alone have lower activity than that the total osmeterium secretion from the Asiatic Troidini Atrophaneura alcinous, which also contains sesquiterpenes and a complex mixture of more polar components, possibly sequestered from the host plant (Aristolochia debilis).  Rejection by birds of aposematic adult Troidini whose larvae feed on Aristolochia was described 35 years ago38,41,53 and aristolochic acids were found in several members of this tribe87-89. Chicks and ants also taste-rejected the aposematic larvae of the swallowtail butterfly Battus polydamas, but other invertebrate predators such as the reduviid bugs Arilus sp. and Montina confusa did not90. It is interesting to note that Aristolochia plants have other nitrophenanthrene derivatives, such as aristolactams (15) and benzoisoquinoline alkaloids (16)84, that have not yet been tested.Glycosidase inhibitorsGlycosidase inhibitors are widespread in plants and can be sequestered by Lepidoptera, for whom they probably serve for defense by making the insects indigestible to a range of potential predators91,92. A very interesting

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