Sequestration of ingested [14C]senecionineN-oxide in the exocrine defensive secretions of chrysomelid beetles

Oreina cacaliae (Chrysomelidae) sequesters in its elytral and pronotal defensive secretion theN-oxides of pyrrolizidine alkaloids (PAN-oxides) from its food plantAdenostyles alliariae (Asteraceae). [¹⁴C]SenecionineN-oxide was applied for detailed studies of PAN-oxide sequestration. An average of 11.4% of total radioactivity is taken up by individual beetles which had received [¹⁴C]senecionineN-oxide with their food leaves 8 days before. An average of 28.9% of the ingested radioactivity could be recovered from the defensive secretions collected twice, i.e., 5 and 8 days after tracer feeding. The tracer transfer into the secretion seems to be a slow but progressive process as indicated by the high percentage of tracer still recovered from the secretion sampled after 8 days. Chromatographic analysis revealed that [¹⁴C]senecionineN-oxide is the only labeled compound in the defensive secretion. Beetles that fed on tertiary [¹⁴C]senecionine sequestered only trace amounts of radioactivity (exclusively present as labeled IV-oxide) in their secretions.O. speciosissima, a species also adapted to PA containing food plants, was shown to sequester [¹⁴C]senecionineN-oxide with the same efficiency asO. cacaliae. O. bifrons, a specialist feeding onChaerophyllum hirsutum (Apiaceae), rejected PA treated leaf samples already at very low PA concentrations (10 nmol/leaf piece). In bothO. cacaliae andO. speciosissima, [¹⁴C]senecionineN-oxide applied by injection into the hemolymph is rapidly transferred into the glands.O. bifrons, not adapted to pyrrolizidine alkaloid containing plants was unable to sequester [¹⁴C]-senecionineN- oxide in the secretion but rapidly eliminated the tracer with the frass. Again, only traces of labeled [¹⁴C]senecionineN-oxide were found in the defensive secretions of the two PA adapted species if labeled senecionine was injected. It is suggested that the beetles are adapted to theN-oxide form of PAs, similarly as their food plants, and that they lack the ability to efficientlyN-oxidize tertiary PAs. No indication forde novo PA synthesis by the beetles was found in tracer feeding experiments with the biogenetic PA precursor putrescine.     

Distribution of autogenous and host-derived chemical defenses inOreina leaf beetles (Coleoptera: Chrysomelidae)

The pronotal and elytral defensive secretions of 10Oreina species were analyzed. Species feeding on Apiaceae, i.e.,O. frigida andO. viridis, or on Cardueae (Asteraceae), i.e.,O. bidentata, O. coerulea, andO. virgulata, produce species-specific complex mixtures of autogenous cardenolides.O. melanocephala, which feeds onDoronicum clusii (Senecioneae, Asteraceae), devoid of pyrrolizidine alkaloids (PAs) in its leaves, secretes, at best, traces of cardenolides. Sequestration of host-plant PAs was observed in all the other species when feeding on Senecioneae containing these alkaloids in their leaves.O. cacaliae is the only species that secretes host-derived PA N-oxides and no autogenous cardenolides. Differences were observed in the secretions of specimens collected in various localities, because of local differences in the vegetation. The other species, such asO. elongata, O. intricata, andO. speciosissima, have a mixed defensive strategy and are able both to synthesize de novo cardenolides and to sequester plant PA N-oxides. This allows a great flexibility in defense, especially inO. elongata andO. speciosissima, which feed on both PA and non-PA plants. Populations of these species were found exclusively producing cardenolides, or exclusively sequestering PA N-oxides, or still doing both, depending on the local availability of food-plants. Differences were observed between species in their ability to sequester different plant PA N-oxides and to transform them. Therefore sympatric species demonstrate differences in the composition of their host-derived secretions, also resulting from differences in host-plant preference. Finally, within-population individual differences were observed because of local plant heterogeneity in PAs. To some extent these intrapopulation variations in chemical defense are tempered by mixing diet and by the long-term storage of PA N-oxides in the insect body that are used to refill the defensive glands.     

Sequestration and metabolism of protoxic pyrrolizidine alkaloids by larvae of the leaf beetle Platyphora boucardi and their transfer via pupae into defensive secretions of adults

Several neotropical leaf-beetles of the genus Platyphora ingest and specifically metabolize plant acquired pyrrolizidine alkaloids (PAs) of the lycopsamine type (e.g., rinderine or intermedine) and enrich the processed alkaloids in their exocrine defensive secretions. In contrast to the related palaearctic leaf beetles of the genus Oreina, which absorb and store only the non-toxic alkaloid N-oxides, Platyphora sequesters PAs exclusively as protoxic tertiary amines. In this study, the ability of P. boucardi larvae to accumulate PAs was investigated. Tracer studies with [¹⁴C]rinderine and its N-oxide revealed that P. boucardi larvae, like adult beetles, utilize the two alkaloidal forms with the same efficiency, but accumulate the alkaloid as a tertiary amine exclusively. Ingested rinderine is rapidly epimerized to intermedine, which is localized in the hemolymph and all other tissues; it is also detected on the larval surface. Like adults, larvae are able to synthesize their own alkaloid esters (beetle PAs) from orally administered [¹⁴C]retronecine and endogenous aliphatic 2-hydroxy acids. These retronecine esters show the same tissue distribution as intermedine. A long-term feeding experiment lasting for almost four months revealed that retronecine esters synthesized from [¹⁴C]retronecine in the larvae are transferred from larvae via pupae into the exocrine glands of adult beetles. Pupae contain ca. 45% of the labeled retronecine originally ingested, metabolized, and stored by larvae; ca. 12% of larval radioactivity could be recovered from the defensive secretions of adults sampled successively over two and a half months. Almost all of this radioactivity is found in the insect-made retronecine esters that are highly enriched in the defensive secretions, i.e., more than 200-fold higher concentration compared to pupae.     

Direct Evidence for Membrane Transport of Host-Plant-Derived Pyrrolizidine Alkaloid N-Oxides in Two Leaf Beetle Genera

The chrysomelid leaf beetles Longitarsus jacobaeae, Oreina cacaliae, and O. speciosissima sequester pyrrolizidine alkaloids from their asteracean host plants and store them as nontoxic N-oxides. Previous analyses showed that Longitarsus is able to N-oxidize protoxic tertiary PAs, but did not resolve in which form N-oxides are taken up. For Oreina, beetles seem able to directly transmit the polar PA N-oxides from the gut into the hemolymph and prevent any reduction of them in the gut yielding protoxic free bases. Here, we confirm the predicted direct uptake of PAs as N-oxides by Oreina, and elucidate the situation for Longitarsus by applying double-labeled [14C]senecionine [18O]N-oxide as tracer. The beetles were fed with the tracer and subsequently senecionine N-oxide was recovered from the defensive secretions (Oreina) and beetle extracts (Longitarsus), purified by HPLC, and submitted to ESI-MS, GC-MS, and analysis of the specific radioactivity. The 18O-label is retained without any loss in the labeled senecionine N-oxide recovered from the two Oreina species. Analysis of the Longitarsus experiment was complicated by a contamination of the HPLC-purified senecionine N-oxide with a second compound, identified as a dihydrosenecionine N-oxide by high-resolution CID analysis. The dihydrosenecionine N-oxide, probably the 15,20-dihydro derivative, constitutes a major idiosyncratic senecionine metabolite present in the beetle. The recovered senecionine N-oxide retained 74% 18O-label. The remaining 25% is mostly due to loss of 18O by reduction and subsequent re-N-oxidation. The experiments confirm for both beetle genera a direct uptake of the polar nontoxic PA N-oxides, which requires specific membrane carriers. Accumulation of detrimental free base PA is prevented by glucosylation (Oreina) or N-oxidation (Longitarsus).     

Production of cardenolides versus sequestration of pyrrolizidine alkaloids in larvae ofOreina species (Coleoptera,Chrysomelidae)

Adult leaf beetles of the genusOreina are known to be defended either by autogenously produced cardenolides or by pyrrolizidine alkaloids (PAs) sequestered from the food plant, or both. In this paper we analyze larvae of differentOreina species and show that the larvae contain the same defensive toxins as the adults in quantities similar to those released in the adults' secretion. Both classes of toxins are found in the body and hemolymph of the larvae, despite their different origins and later distribution in the adults. Larvae of sequestering species differed in their PA patterns, even though they fed on the same food plants. The concentration in first-instar larvae of a PA-sequestering species was similar to that in fourth-instar larvae. In all stages examined, the amount of PAs per larva did not greatly exceed the estimated uptake of one day. Eggs of two oviparous species contained large concentrations of the adult's toxins, while neonates of a sequestering larviparous species had no PAs.     

Taste receptors for pyrrolizidine alkaloids in a monophagous caterpillar

Caterpillars of Utetheisa ornatrix are monophagous on species of Crotalaria from which they obtain pyrrolizidine alkaloids (PAs) for defense and which the males convert to a pheromone. We show that a taste receptor neuron in each of the lateral and medial galeal styloconic sensilla responds to PAs of three different types. Monocrotaline, commonly present in Crotalaria species, is the most strongly stimulating with thresholds of response below 10^–11 and 10^–9 M in the two sensilla. These are among the lowest known taste thresholds in any insect and are similar to the thresholds for PAs in a polyphagous arctiid caterpillar that also sequesters PAs and uses them as the source of male pheromone. The receptors also respond to heliotrine, a type of PA that is probably never encountered by the insects. Monocrotaline and senecionine N-oxide are shown in behavioral assays to be phagostimulants. The data show that there is no tight link between taste receptor sensitivity to specific PAs and hostplant selection in these caterpillars. Perhaps the adults are primarily responsible for hostplant selection.     

Mate choice and toxicity in two species of leaf beetles with different types of chemical defense

Evidence for the use of defensive compounds for sexual purposes is scarce, even though sexual selection might have some importance for the evolution of defensive traits. This study investigates the effect of defense-related traits and body size on mating success in two sister species of leaf beetle differing in their type of chemical defense. Oreina gloriosa produces autogenous cardenolides, whereas O. cacaliae sequesters pyrrolizidine alkaloids from its food plant. Larger O. gloriosa males with more toxin or higher toxin concentration had a mating advantage, likely due to direct or indirect female choice. In the laboratory, particular pairings recurred repeatedly in this species, indicating mate fidelity. O. gloriosa females were also subject to sexual selection, possibly by male choice, because larger females and those with higher toxin concentration mated more readily and more often. In O. cacaliae, in contrast, sexual selection for toxicity and body size was not detected, or at best was much weaker. Because toxicity is heritable in O. gloriosa but environment-dependent in O. cacaliae, individuals of the former species could be choosing well-defended partners with good genes. Our study suggests that sexual selection may contribute to the maintenance of heritable defensive traits.     

Acquired and Partially De Novo Synthesized Pyrrolizidine Alkaloids in Two Polyphagous Arctiids and the Alkaloid Profiles of Their Larval Food-Plants

The profiles of pyrrolizidine alkaloids (PAs) in the two highly polyphagous arctiids Estigmene acrea and Grammia geneura and their potential PA sources in southeastern Arizona were compiled. One of four species of Boraginaceae, Plagiobothrys arizonicus, contained PAs; this is the first PA record for this plant species. The principle PA sources are Senecio longilobus (Asteraceae) and Crotalaria pumila (Fabaceae). The known PA pattern of S. longilobus was extended; the species was found to contain six closely related PAs of the senecionine type. Three novel PAs of the monocrotaline type, named pumilines A-C, were isolated and characterized from C. pumila, a species not studied before. The pumilines are the major PAs in the seeds, while in the vegetative organs they are accompanied by the simple necine derivatives supinidine and as the dominant compound subulacine (1beta,2beta-epoxytrachelanthamidine). In both plant species, the PAs are stored as N-oxides, except C. pumila seeds, which accumulate the free bases. Great variation in PA composition was observed between local populations of C. pumila. The PA profiles were established for larvae and adults of E. acrea that as larvae had fed on an artificial diet supplemented with crotalaria-powder and of G. geneura fed with S. longilobus. In both experiments, the larvae had a free choice between the respective PA source and diet or food plants free of PAs. The profiles compiled for the two species reflect the alkaloid profiles of their PA sources with one exception, subulacine could never be detected in E. acrea. Besides acquired PAs, insect PAs synthesized from acquired necine bases and necic acids of insect origin were detected in the two arctiid species. These insect PAs that do not occur in the larval food sources accounted for some 40-70% (E. acrea) and 17-37% (G. geneura) of total PAs extracted from the insects. A number of novel insect PAs were identified. Plant-acquired and insect PAs were found to accumulate as N-oxides. The results are discussed in relation to specific biochemical, electrophysiological, and behavioral mechanisms involved in PA sequestration by arctiids.     

Specialist Weevil, Rhyssomatus lineaticollis, Does Not Spatially Avoid Cardenolide Defenses of Common Milkweed by Ovipositing into Pith Tissue

Rhyssomatus lineaticollis is a milkweed specialist whose larvae feed upon pith parenchyma in ramet stems of the common milkweed, Asclepias syriaca. Compared with other specialist insect herbivores on milkweeds, this curculionid beetle is unusual in that it is cryptically colored and does not sequester cardenolides characteristic of milkweed chemical defense. Based upon optimal defense theory, we predicted that pith tissue would be low in defensive compounds and that oviposition into the pith would spatially avoid cardenolides. We rejected this hypothesis because we found that pith tissue has a relatively high cardenolide concentration compared to cortex, epidermis, and leaf tissues. Moreover, we found total plant cardenolide concentration was lower in plants that contained the beetle eggs. Cardenolide concentrations were different among tissues in intact stems without the pith herbivore compared to stems where it was present. Furthermore, the overall polarity of the cardenolides present varied among plant tissues and between plants with and without R. lineaticollis eggs. Although we found lower concentrations of cardenolide in piths where the eggs were present, the cardenolides present in the pith contained more nonpolar forms, indicating that the plant may be responding to herbivory by increasing toxic efficacy of cardenolide defenses while lowering the total concentration. We suggest that preoviposition behavior by female beetles, which includes feeding on new leaves of the plant, is a mechanism by which females manipulate plant chemistry and assess quantitative and qualitative changes in cardenolide chemistry in response to herbivory prior to oviposition.     

Palatability of aposematic queen butterflies (Danaus gilippus) feeding onSarcostemma clausum (Asclepiadaceae) in Florida

Queen butterflies (Danaus gilippus) are generally considered unpalatable to predators because they sequester and store toxic cardenolides from their larval food plants. However, a major queen food plant in Florida, the asclepiadaceous vineSarcostemma clausum, is shown here to be a very poor cardenolide source, and queens reared on this plant contain no detectable cardenolide. A direct evaluation of queen palatability using red-winged blackbirds indicates thatS. clausum-reared butterflies are essentially palatable to these predators (85% of abdomens entirely eaten), indicating little protection from either cardenolides, other sequestered phytochemicals, or de novo defensive compounds. Wild-caught queens that presumably fed as larvae uponS. clausum and also had access to adult-obtained chemicals, such as pyrrolizidine alkaloids (PAs), were relatively palatable as well (77% of abdomens eaten); they did not differ significantly in palatability from the labreared butterflies. Together, these findings suggest that; (1)S. clausumfed queens are poorly defended against some avian predators, and (2) for the particular queen sample examined, PAs do not contribute substantially to unpalatability. The discovery thatS. clausum-feeding queens are essentially palatable is of additional significance because it compels a reassessment of the classic mimicry relationship between queen and viceroy butterflies. Viceroys have been shown recently to be moderately unpalatable; therefore, the established roles of model and mimic may be reversed in some cases.     

Production of cardiac glycosides by chrysomelid beetles and larvae

Cardenolides were looked for in 17 chrysomelid beetles belonging to 11 genera from three subfamilies, and they were found only inChrysolina andChrysochloa species (Chrysomelinae, Chrysolinini). The food plants of these insects are not known to produce cardenolides. TheChrysochloa and mostChrysolina species secrete a complex mixture of cardenolides, butChrysolina didymata secretes a single compound, andChrysolina carnifex, none. Several quantitative and perhaps qualitative differences were observed in the patterns of cardenolides produced by far distant populations of bothChrysolina polita andC. herbacea, collected in either France and Belgium, or Greece. These differences remain constant from one generation to the other, whatever the food plant is, and appear to be genetic. InC. polita from Greece, the pattern is unchanged after four generations bred in the laboratory onMentha ×villosa, which is known to be without cardenolides. In adults, the cardenolides are released with the secretion of the pronotal and elytral defensive glands, but in the larvae which lack the defensive glands, cardenolides are also produced. The total amount of cardenolides and the complexity of their mixture increases through the life cycle of the insects. The six main cardenolides secreted byC. coerulans were identified as: sarmentogenin, periplogenin, bipindogenin, and their corresponding xylosides.C. didymata secretes only sarmentogenin.     

Male sex pheromone of a giant danaine butterfly,Idea leuconoe

Males of a giant danaine butterfly,Idea leuconoe, display hairpencils during courtship. The females were visually attracted to and olfactorily arrested by an artificial butterfly model to which male hairpencil extracts were added. The hairpencil extracts contained a complex mixture of volatiles, including pyrrolizidine alkaloid (PA) derivatives (danaidone, viridifloric -lactone), aromatics (phenol,p-cresol, benzoic acid), terpenoids (geranyl methyl thioether, (E,E)-farnesol), a series of -lactones (6-hydroxy-4-undecanolides and its homologs), hydrocarbons [(Z)-9-tricosene, etc.], and several compounds with higher molecular weight. A mixture of the major volatiles applied to a butterfly dummy strongly elicited an abdomen-curling acceptance posture in females. Viridifloric -lactone and danaidone induced significant electroantennogram responses on the female's antennae, suggesting their principal role together with other hairpencil components as a sex pheromone to seduce females.I. leuconoe males seem to acquire the precursor for both of the PA fragments from the host plant,Parsonsia laevigata (Apocynaceae), during the larval stage; thereby they do not show pharmacophagous behavior towards PA-containing plants during the adult stage. However, males are pharmacophagously attracted to and feed on a number of simple phenolic compounds in a manner similar to other danaine species towards PAs. Wild males sequester one of the phagostimulants, (–)-mellein, in the hairpencils in varying quantities. Phenolic compounds incorporated in the hairpencils may act primarily as warning odors linked with the defensive PAs present in the body tissues.     

Effect of urea and salt on micelle formation of zwitterionic surfactants

The effect of urea on micelle formation of zwitterionic surfactants was investigated by measuring conductivity, critical micelle concentration (CMC), relative viscosity, and the spectrophotometric shift in wavelength. We examined two zwitterionic surfactants, N,N-dimethyl dodecylamine N-oxide and N,N-dimethyl tetradecylamine N-oxide (DMTAO). The CMC values of the surfactants increased with the addition of urea. Also, the relative viscosity of the surfactant solutions decreased at higher concentrations of urea and increased with increasing KCl concentration. The absorbance maxima of the surfactants decreased with increasing urea concentration.     

Chemical basis of egg cannibalism in a caterpillar (Utetheisa ornatrix)

Larvae of the mothUtetheisa ornatrix are shown to cannibalize eggs in the laboratory. They proved most cannibalistic if they were systemically deficient in pyrrolizidine alkaloid (PA), the defensive agent that protectsUtetheisa at all stages of development against predation, and whichUtetheisa acquire as larvae from their food plant. In exercising cannibalistic choice,Utetheisa larvae feed preferentially on eggs that are PA-Iaden rather than PA-free. Egg cannibalism can therefore provideUtetheisa with a supplemental means of PA procurement. Moreover, presence of PA in the egg, while providing the egg with defense against predation, can increase its vulnerability to cannibalism. Although evidence is presented thatUtetheisa larvae cannibalize eggs in nature, it is argued that such feeding may occur only opportunistically in the wild, rather than as a matter of course.Paper No. 104 of the series Defense Mechanisms of Arthropods, No. 103 in Eisner and Eisner, 1991.Psyche 98:111–118.     

Are Insect-Synthesized Retronecine Esters (Creatonotines) the Precursors of the Male Courtship Pheromone in the Arctiid Moth Estigmene acrea?

The pyrrolizidine alkaloid (PA) profiles were determined for adults of the polyphagous arctiid Estigmene acrea, which as larvae had fed on artificial diet supplemented with Crotalaria-pumila powder with known concentrations of PAs. The larvae always had a free choice between alkaloid-containing and plain diets. The alkaloid profiles of adults revealed a striking sexual dimorphism. Both sexes contained macrocyclic PAs of the monocrotaline type sequestered from the diet and, in addition, a substantial proportion of supinidine and retronecine esters synthesized by the insects from necine bases derived from the dietary alkaloids and necic acids of insect origin. These insect alkaloids accounted for 35% and 55% of total PAs in males and females, respectively. The difference was that in females the retronecine esters (creatonotines) made up 58 g (43% of total PAs), while males contained a fivefold lower proportion, 12 g (13%). Four of the ten male individuals analyzed were found devoid of creatonotines. Based on the experimental data in combination with evidence from the literature, it is suggested that the creatonotines are direct pheromone precursors in E. acrea. It is hypothesized that this may represent a general mechanism of hydroxydanaidal formation from diverse macrocyclic PAs in arctiids.     

Selective Sequestration of Pyrrolizidine Alkaloids from Diverse Host Plants by Longitarsus Flea Beetles

In 11 species of the flea beetle genus Longitarsus we investigated whether the insects sequester the pyrrolizidine alkaloids (PAs) present in their host plants of the families Asteraceae and Boraginaceae. In all cases where PAs could be detected in the leaves of the local host plant, they could also be detected in the corresponding beetles. In one host plant, Pulmonaria officinalis, no PAs could be detected in the leaves, yet were present in the beetles collected from them. We suggest this is due to uptake of PAs during the root-feeding larval stage. By comparing the GC-MS pattern of PAs found in the beetles with those of their hosts, we investigated the specificity of this sequestration. Furthermore, we compared the pattern of sequestered PAs across beetle species that had been feeding on the same plant, and across hosts in Longitarsus species that feed on different plants in the field. This allowed us to analyze to what extent the PA pattern in the insects is specific for the beetle species and depends on the local food plant. Our data indicate that the PAs found in the beetles are largely determined by the host plant, e.g., whether alkaloids typical of the Boraginaceae or Asteraceae are present. However, there are some indications for a selective uptake of PAs and apparently the beetles are able to metabolize them.     

Sequestration ofVeratrum alkaloids by specialistRhadinoceraea nodicornis konow (Hymenoptera,Tenthredinidae) and its ecoethological implications

The larvae of the specialist sawflyRhadinoceraea nodicornis Konow (Hymenoptera, Tenthredinidae) store in their hemolymph ceveratrum alkaloids originating from the host plantVeratrum album L. (Liliales, Melanthiaceae). The major alkaloid found in the hemolymph is 3-acetyl-zygadenine. Qualitative and quantitative data showed that the plant alkaloid 3-angeloylzygadenine is most probably metabolized in the larval gut to zygadenine and then acetylated. A still unidentified alkaloid with a molecular weight of 591 Da was detected in plant leaves as well as in the gut, hemolymph, and excrement of larvae. Protoveratrine A and B, on the other hand, seem to be degraded by the larvae. These findings indicate that the pathway of ceveratrum alkaloids inR. nodicornis larvae is fourfold: direct sequestration, metabolism followed by sequestration, excretion of intact alkaloids, and degradation. In contrast, no ceveratrum alkaloids were detected in the hemolymph and excrement of larvae of the generalist sawflyAglaostigma sp. fed withV. album leaves. Bioassays with the antMyrmica rubra L. proved that the hemolymph ofR. nodicornis larvae is highly deterrent and toxic. In bioassays evaluating defensive efficiency against predators (ants, spiders, and bushcrickets), no larvae were eaten. Ceveratrum alkaloids were also detected in the hibernating prepupae ofR. nodicornis. In feeding bioassays, the shrewCrocidura russula Hermann rarely fed upon prepupae, suggesting that this stage is also protected from predation to some degree. In field surveys, the only parasitoids recorded were two ichneumonid species that are believed to be specialized onR. nodicornis. Bioassays and field observations enable us to suppose thatR. nodicornis and its enemies produce a food web of ion connectance.     

Colorado Potato Beetle Toxins Revisited: Evidence the Beetle Does Not Sequester Host Plant Glycoalkaloids

The Colorado potato beetle feeds only on glycoalkaloid-laden solanaceous plants, appears to be toxic to predators, and has aposematic coloration, suggesting the beetle may sequester alkaloids from its host plants. This study tested 4th instars and adults, as well as isolated hemolymph and excrement, to determine if the beetles sequester, metabolize, or excrete alkaloids ingested from their host plants. HPLC analysis showed: that neither the larvae nor the adults sequestered either solanine or chaconine from potato foliage; that any alkaloids in the beetles were at concentrations well below 1 ppm; and that alkaloids were found in the excrement of larvae at approximately the same concentrations as in foliage. Analysis of alkaloids in the remains of fed-upon leaflet halves plus excreta during 24 hr feeding by 4th instars, as compared to alkaloids in the uneaten halves of the leaflets, showed that equal amounts of alkaloids were excreted as were ingested. The aposematic coloration probably warns of a previously-identified toxic dipeptide instead of a plant-derived alkaloid, as the Colorado potato beetle appears to excrete, rather than sequester or metabolize, the alkaloids from its host plants.     

Effect of hypoxanthine-3(N)-oxide and hypoxanthine-1(N)-oxide on central nervous excitation of the black tetraGymnocorymbus ternetzi (Characidae,Ostariophysi, Pisces) indicated by dorsal light response

The change of state in the central nervous system ofGymnocorymbus ternetzi after detection of hypoxanthine-l(N)-oxide, hypoxanthine-3(N)-oxide, and of the alarm substance from conspecifics was measured quantitatively by means of the fishes' equilibrium behavior. The fish swam freely in a tiny cage, illuminated horizontally from one side. The change of the angle of inclination of the dorsoventral axis of the fish was registered by means of a videorecorder. The recordings were later measured on the monitor in single frames at 0.2-sec intervals where the equilibrium position of the fish could be accurately determined ± 1 °. Various substances were presented to the fish, and their effects upon equilibrium position were recorded. An enhanced optical alertness shown by an increase in the fishes' inclination was generally produced with alarm substance. Without any additional stimulation, the factorU, representing quantitatively the degree of the change of central state, varied slightly within the experimental period of 1 min; however, this factor never exceededU= 1.0 ± 0.15 in control fish. The increase ofU usually exceeded considerably the value 1.15 when skin extract from conspecifics or 7–8 g of hypoxanthine-3(N)-oxide were given. However, when hypoxanthine-l (N)-oxide was presented,U generally did not exceed 1.15. The difference between hypoxanthine-3(N)-oxide and hypoxanthine-l(N)-oxide was highly significant. This result is in accordance with the findings on fish schools ofDanio malabaricus, where hypoxanthine-3(N)-oxide elicited the fright reaction, but hypoxanthine-1(N)-oxide was ineffective. The results support the hypothesis that the alarm substance from the skin ofPhoxinus phoxinus is identical with hypoxanthine-3(N)-oxide. The results with alarm substance or hypoxanthine-3(N)-oxide did not show any adaptation. This was also true in fish that were stimulated repeatedly at intervals of a couple of minutes only. InGymnocorymbus, which has compensated for removal of the otolith of one utriculus, conspecific skin extract triggers the typical postoperative phenomenon, i.e., rotation around the fishes' long axis towards the operated side. Whereas such a decompensation could be elicited by hypoxanthine-3(N)-oxide as well, hypoxanthine-l(N)-oxide had no effect. This finding is interpreted as an effect of the alarm substance and of hypoxanthine-3(N)-oxide on the centers of equilibrium.     

Biparental Endowment of Endogenous Defensive Alkaloids in <Emphasis Type="Italic">Epilachna paenulata</Emphasis>

Coccinellid beetles contain a variety of defensive alkaloids that render them unpalatable to predators. Epilachna paenulata (Coleoptera: Coccinellidae) is a South American ladybird beetle that feeds on plants of the Cucurbitaceae family. The defensive chemistry of E. paenulata has been characterized as a mixture of systemic piperidine, homotropane, and pyrrolidine alkaloids. Whole body extracts of adult beetles contain four major alkaloids: 2-(2′-oxopropyl)-6-methylpiperidine (1); 1-(6-methyl-2,3,4,5-tetrahydro-pyridin-2-yl)-propan-2-one (2); 1-methyl-9-azabicyclo[3.3.1]nonan-3-one (3); and 1-(2″-hydroxyethyl)-2-(12′-aminotridecyl)-pyrrolidine (4). Comparative studies of the defensive chemistry of eggs, larvae, pupae, and adults showed differences in alkaloid composition and concentration among life stages. While adults contained mainly the homotropane 1-methyl-9-azabicyclo[3.3.1]nonan-3-one (3), eggs showed the highest concentration of the piperidine 2-(2′-oxopropyl)-6-methylpiperidine (1). We studied the origin of this alkaloid in the eggs by feeding newly emerged, virgin adult beetles with [2-¹³C]-labeled acetate, and by performing crosses between ¹³C-fed and unlabeled males and females. GC-MS analysis of alkaloids from ¹³C-fed males and females showed high incorporation of ¹³C into the alkaloids, as evidenced from a 20–30% increase of isotopic peaks in diagnostic fragment ions, confirming the expected endogenous origin of these alkaloids. In addition, analyses of eggs from different crosses showed that labeled alkaloids from both parents are incorporated into eggs, indicating that E. paenulata males transfer alkaloids to the females at mating. Biparental endowment of chemical defenses into eggs has been shown previously in insects that acquire defensive compounds from dietary sources. To our knowledge, this is the first report of biparental egg endowment of endogenous defenses. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.