Survival of first instar larvae ofDanaus plexippus (Lepidoptera: Danainae) in relation to cardiac glycoside and latex content ofAsclepias humistrata (Asclepiadaceae)

Summary Our paper addresses field survivorship of first instar monarch butterfly larvae (Danaus plexippus L., Lep.: Danainae) in relation to the dual cardenolide and latex chemical defenses of the sand hill milkweed plant,Asclepias humistrata (Asclepiadaceae) growing naturally in north central Florida. Survival of first instar larvae in the field was 11.5% in the first experiment (15–20 April 1990), and dropped to 3.4% in the second experiment (20–30 April). About 30% of the larvae were found glued to the leaf surface by the milkweed latex. Predator exclusion of non-flying inverte-brates by applying tanglefoot to the plant stems suggested that the balance of the mortality was due to volant inverte-brates, or to falling and/or moving off the plants. Regression analyses to isolate some of the other variables affecting survivorship indicated that first instar mortality was correlated with (1) increasing cardiac glycoside concentration of the leaves, (2) increasing age of the plants, and (3) the temporal increase in concentration of cardiac glycosides in the leaves. The study also provided confirmatory data of previous studies that wild monarch females tend to oviposit onA. humistrata plants containing intermediate concentrations of cardiac glycosides. Cardiac glycoside concentration in the leaves was not correlated with that in the latex. The concentration of cardenolide in the latex is extremely high, constituting an average of 1.2 and 9.5% of the mass of the wet and dry latex, respectively. The data suggest that an increase in water content of the latex is compensated for by an influx of cardenolide with the result that the cardenolide concentration remains constant in the latex systems of plants that are growing naturally. We also observed first instar larvae taking their first bite of milkweed leaves in the field. In addition to confirming other workers findings that monarch larvae possess elaborate sabotaging behaviour of the milkweed's latex system, we discovered that several larvae on their first bite involuntarily imbided a small globule of latex and instantly became cataleptic. This catalepsis, lasting up to 10 min, may have been in response to the high concentration of cardenolide present in the latex ofA. humistrata, more than 10 times that in the leaves. The results of the present study suggest that more attention should be directed to plant chemical defenses upon initial attack by first instar insect larvae, rather than attempting correlations of plant chemistry with older larvae that have already passed the early instar gauntlet. The first bite of neonate insects may be the most critical moment for coping with the chemical defenses of many plants and may play a much more important role in the evolution of insect herbivory than has previously been recognized.     

Responses of three mouse species to deterrent chemicals in the monarch butterfly. II. Taste tests using intact monarchs

Summary Peromyscus melanotis is the only one of three mouse species that eats monarch butterflies at their overwintering sites in Mexico. I tested two hypotheses: 1)P. aztecus avoids monarchs because of a bitter taste aversion to cardiac glycosides (CGs) and an inability to reject CG-rich body parts; 2)Reithrodontomys sumichrasti avoids monarchs principally because of a bitter taste aversion to the CGs. None of the species are sensitive to the toxic effects of ingested CGs. Feeding responses of laboratory-reared mice of each species to monarchs with low, medium and high CG concentrations were compared. BothP. aztecus andR. sumichrasti ate significantly fewer of all three types of monarchs thanP. melanotis. ForP. aztecus andR. sumichrasti, the number of monarchs eaten decreased with increasing CG concentration, whereas forP. melanotis, the number remained constant.Peromyscus melanotis andR. sumichrasti developed a feeding technique for rejecting the CG-laden cuticular material, which reduced the bitterness of ingested monarch material. However,R. sumichrasti displayed the technique significantly less often thanP. melanotis; andP. aztecus never developed it. I conclude that high taste sensitivity to CGs and less versatile food handling preventP. aztecus andR. sumichrasti from overcoming the monarch's chemical defenses.     

Chemical defence in chewing and sucking insect herbivores: Plant-derived cardenolides in the monarch butterfly and oleander aphid

Summary Cardenolide sequestration by a hemimetabolous aphid and a holometabolous butterfly from the neotropical milkweed,Asclepias curassavica L., is compared. The oleander aphid,Aphis nerii B. de F., sequestered a similarly narrow range of cardenolide concentrations to the monarch butterfly,Danaus plexippus (L.), from the wide range of concentrations available in leaves of A.curassavica. However, A.nerii sequestered significantly less cardenolide (269 µg/0.1 g) thanD. plexippus (528 µg/0.1 g). The honeydew excreted by A.nerii was comprised of 46% cardenolide. The complete polarity range of 25 cardenolides detected by thin layer chromatography in A.curassavica was represented in the 17 whole aphid cardenolides and the 20 aphid honeydew cardenolides detected. D.plexippus sequestered a narrower polarity range of 11 cardenolides, having eliminated low polarity cardenolide genins and glycosides. It is suggested that these chemical differences may be related to interactions among the broad feeding tactics of sucking or chewing milkweed leaves, life history constraints of holometabolyversus hemimetaboly, the distribution of milkweed food resources in space and time, and the dynamics of natural enemies.     

Multi-level complexity in the use of plant allelochemicals by aposematic insects

Summary As recognized by Miriam Rothschild as early as the 1960s and repeatedly emphasized in her papers, the use, misuse, or non-use of plant allelochemicals by insects is extremely variable and difficult to predict, at many levels of time, space, and biological organization. Although certain patterns that reoccur have been important in the development of ecological theory, the optimization of cost-benefit equations involving two or three trophic levels, each with large numbers of individuals, populations, and species in erratic and complex interactions, produces unexpected and fascinating scenarios. The development of rapid colorimetric and chromatographic analyses for several types of plant allelochemicals, notably certain groups of alkaloids, cardiac and cyanogenic glycosides, phenolics, terpenes, and glucosinolates, has permitted a detailed investigation of the variation and flow of these substances in natural organisms and ecosystems. The results of these analyses, in our hands mostly for pyrrolizidine alkaloids (PAs), do not suggest a straightforward classical choice by the aposematic insect to simply sequester or synthesize its defences. Rather, they reveal a confusing variety of diffuse and complex patterns that become increasingly closer to chaos as they are multiplied across structures, species, sexes, stages, sites, seasons, and selective regimes. We present a model reflecting results of analyses at this chemoecological interface. Depending upon an initial option, involving the recognition (or not) of a plant allelochemical, the herbivore will face thereafter options to ingest it (or not), and then to tolerate and absorb (or detoxify and excrete), modify (or not), passively, actively or selectively accumulate, turn over (or not), distribute (or concentrate), and use this compound in a variety of growth, defense, or reproductive functions. The herbivore can also quantitatively or qualitatively regulate the intensity or dispersion of its attack on the plant tissues, in order to modify feedback loops of selection on the plant and its chemicals which exist in most of the earlier steps, or those with its predators and parasites that occur in the later ones. Options that lead to mutualism through positive feedback loops will tend to accumulate and become rapidly fixed by natural selection. Additional variations and anomalies such as automimicry, chemical mimicry, sexual dimorphism and communication, selective sequestration and passing-up of allelochemicals, special glands and structures, and synergism effects, are among the secondary complications of this model that have occupied much thought, time, experimental labor, and polemical space in chemical ecology journals and meetings. Examination of the tendencies and results at various points in the model can be used to explain these features and to make further predictions, plan experiments, and devise activity-based bioassays and new chemical analyses. These may lead some day to new and more robust visions of the major patterns of chemical transfer at this widespread and important natural interface.     

Milkweeds,monarch butterflies and the ecological significance of cardenolides

Summary The contribution of Miriam Rothschild to the monarch cardenolide story is reviewed in the light of the 1914 challenge by the evolutionary biologist, E.B. Poulton for North American chemists to explain the chemical basis of unpalatability in monarch butterflies and their milkweed host plants. This challenge had lain unaccepted for nearly 50 years until Miriam Rothschild took up the gauntlet and showed with the help of many able colleagues that monarchs are aposematically coloured because they sequester toxic cardenolides from milkweed host plants for use as a defence against predators. By virtue of Dr Rothschild's inspiration and industry, and subsequently that of Lincoln Brower and his colleagues, this tritrophic interaction has become a familiar paradigm for the evolution of chemical defences and warning colouration. We now know that the cardenolide contents of different milkweeds vary quantitatively, qualitatively and spatially, both within and among species and we are starting to appreciate the implications of such variation. However, as Dr Rothschild has pointed out in her publications, cardenolides have sometimes blinded us to reality and it is curious how little evidence there is for a defensive function to cardenolides in plants — especially against adapted specialists such as the monarch. Thus the review will conclude with a discussion of the significance of temporal variation and induction of cardenolide production in plants, the lethal plant defence paradox and an emphasis on the dynamics of the cardenolide-mediated interaction between milkweeds and monarch larvae.     

Responses of three mouse species to deterrent chemicals in the monarch butterfly. I. Taste and toxicity tests using artificial diets laced with digitoxin or monocrotaline

Summary Of three common mouse species at the Mexican overwintering sites of the monarch butterfly, onlyPeromyscus melanotis eats monarchs. We hypothesized thatP. aztecus andReithrodontomys sumichrasti reject monarchs because they are more sensitive to the bitter taste and/or toxic effects of the cardiac glycosides (CGs) and pyrrolizidine alkaloids (PAs) in the butterflies. Two-choice preference tests revealed no difference in taste avoidance thresholds to free base and N-oxide forms of the PA, monocrotaline, but very different avoidance thresholds to the CG, digitoxin. Avoidance thresholds forR. sumichrasti andP. aztecus were, in respective order, 1020 and 34 times less than that forP. melanotis. We also tested the toxic sensitivity of juvenile mice by chronically feeding diets containing digitoxin or monocrotaline at concentrations similar to those used in the preference tests. No species developed CG toxicity, but bothP. melanotis andP. aztecus developed moderate PA toxicity (R. sumichrasti was not tested for PA toxicity).P. aztecus grew more slowly and manyP. melanotis had hepatic metabolic lesions. Thus, the three mouse species responded very differently to the taste and toxic properties of CGs and PAs at ecologically relevant concentrations: 1) CGs were taste rejected by all species exceptP. melanotis, while PAs were not; and 2) PAs were toxic, while CGs were not.     

Molecular studies on the ouabain binding site of the Na+, K+-ATPase in milkweed butterflies

Summary. The Na⁺, K⁺-ATPase of the Monarch butterfly (Danaus plexippus) is insensitive to the inhibition by cardiac glycosides due to an amino acid replacement: histidine instead of asparagine at position 122 of the α-subunit representing the ouabain binding site. By PCR amplification of the DNA sequence of this site, a PCR product of 270 bp was obtained from DNA extracted from Danainae species (Danaus plexippus, D. chrysippus, D. gillipus, D. philene, D. genutia, Tirumala hamata, Euploea spp., Parantica weiskei, P. melusine), Sphingidae (Daphnis nerii) and mimics of milkweed butterflies (Hypolimnas missipus, Limenitis archippus and L. arthemis, Nymphalidae). Analysis of the nucleotide sequences revealed that the single point mutation in the ouabain binding domain (AAC-Asn for CAC-His) was present only in Danaus plexippus, but not in the other species investigated. Since these milkweed butterflies also store cardenolides, other structural modifications of the Na⁺, K⁺-ATPase may have occurred or other strategies of cardenolide tolerance have been developed. Received 15 May 2000; accepted 29 June 2000