Pyrrolizidine alkaloids in the arctiid mothHyalurga syma

The arctiid mothHyalurga syma (subfamily Pericopinae) sequesters pyrrolizidine alkaloids (PAs) from its larval food plantHeliotropium transalpinum (Boraginaceae). Colorimetric quantification of total PAs in the larvae, pupae, and adults ofHyalurga revealed mean values of about 286–445g per individual (1.4–2.6% of dry weight). The PA mixtures found in the moth and its larval food plant were evaluated by GC-MS. Food-piant leaves were found to contain the diastereoisomeric retronecine esters indicine (IIIa), intermedine (IIIb), and lycopsamine (IIIc), and the heliotridine ester rinderine (IIId) only as minor constituents, whereas 3-acetylrinderine (IVc) (68% of total PAs) and the respective 3-acetyl esters of indicine (IVa) and intermedine (IVb) (both 17%) were the major alkaloids. Supinine (IIa) is detectable in traces only. The PA mixtures in eggs, larvae, pupae, and imagines ofHyalurga were identical: indicine, intermedine, and lycopsamine accompanied by considerable amounts of supinine and amabiline or coromandalinine (IIb/IIc) were the major components. Only larvae were found to store small quantities of a 3-acetyl derivative. Rinderine and its 3-acetyl ester were never found in the insects. Low concentrations of the arctiidspecific PA callimorphine (I) were present in larvae, pupae, and imagines. The differences in the PA patterns of the insects and their larval food plant suggest thatHyalurga is capable of modifying plant-derived PAs by inversion of the 7-OH configuration (conversion of the necine base heliotridine into retronecine), and perhaps the inversion of the 3-OH [conversion of (+)-trachelanthic acid into (–)-viridifloric acid], although the possibility of a selective sequestration of the respective retronecine esters cannot be excluded. Some trials with the orb-weaving spiderNephila clavipes, a common neotropical predator, showed that both freshly emerged and field-caught adults ofHyalurga syma are liberated unharmed by the spider. The liberation could be related to the presence of PAs in the moths.     

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.     

Sequestration, Maintenance, and Tissue Distribution of Pyrrolizidine Alkaloid N-Oxides in Larvae of Two Oreina Species

Oreina cacaliae and O. speciosissima are leaf beetles that, as larvae and adults, sequester pyrrolizidine alkaloid N-oxides (PAs) as defensive compounds from their host plants Adenostyles alliariae and Senecio nemorensis. As in most Oreina species, O. speciosissima is also defended by autogenously produced cardenolides (mixed defensive strategy), whereas O. cacaliae does not synthesize cardenolides and is exclusively dependent on host-plant-acquired PAs (host-derived defense). Adults of the two Oreina species were found to have the same PA storage capacity. The larvae, however, differ; larvae of O. speciosissima possess a significantly lower capability to store PAs than O. cacaliae. The ability of Oreina larvae to sequester PAs was studied by using tracer techniques with ¹⁴C-labeled senecionine N-oxide. Larvae of the two species efficiently take up [¹⁴C]senecionine N-oxide from their food plants and store the alkaloid as N-oxide. In O. cacaliae, there is a slow but continuous loss of labeled senecionine N-oxide. This effect may reflect the equilibrium between continuous PA uptake and excretion, resulting in a time-dependent tracer dilution. No noticeable loss of labeled alkaloid is associated with molting. Senecionine N-oxide is detectable in all tissues. The hemolymph is, with ca. 50–60% of total PAs, the major storage compartment, followed by the integument, with ca 30%. The alkaloid concentration in the hemolymph is approximately sixfold higher than in the solid tissues. The selectivity of PA sequestration in larvae is comparable to PA sequestration in the bodies of adult beetles.     

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.     

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.     

Antimicrobial Activity of Exocrine Glandular Secretion of Chrysomela Larvae

The exocrine glandular secretions of leaf beetle larvae of the taxon Chrysomela are well-known defensive devices used against some generalist predators. Salicylaldehyde is the major repellent component of secretions emitted by larvae of Chrysomela vigintipunctata and C. lapponica, which feed on salicin-rich Salicaceae. In this study, we examined whether salicylaldehyde is also active against the entomopathogenic fungus Metarhizium anisopliae. The germination and growth of this fungus was strongly inhibited when salicylaldehyde was applied directly onto the blastospores. The salicylaldehyde concentration of the larval secretions of the tested willow feeding Chrysomela larvae was much higher than the one necessary to display this antifungal activity. Additionally, salicylaldehyde was shown to reduce germination and growth of M. anisopliae via the gas phase over a distance of 45 mm. Further studies on the antimicrobial activity of the salicylaldehyde-containing secretions of Chrysomela larvae revealed that they act nonspecifically against prokaryotic (Escherichia coli) and eukaryotic cells (Saccharomyces cerevisiae and Trichoplusia ni). All antimicrobial and cytotoxic effects detected proved to be due to salicylaldehyde. The larval secretions of the birch-feeding allospecies of C. lapponica, that do not contain salicylaldehyde, but mainly carboxylic acids and their esters, showed no detectable effects on bacteria or fungi and no cytotoxic effects against insect cells. The results are discussed with respect to their ecological relevance.     

Differential Utilization of Pyrrolizidine Alkaloids by Males of a Danaid Butterfly, <Emphasis Type="Italic">Parantica sita</Emphasis>, for the Production of Danaidone in the Alar Scent Organ

Males of the chestnut tiger butterfly, Parantica sita, secrete danaidone as a major component from the alar androconial organ (sex brand). Since danaidone has been postulated to be derived from various pyrrolizidine alkaloids (PAs), which males ingest as adults from PA-containing plants, we conducted oral administration tests of several PAs to examine their availability for danaidone production by P. sita males. Males fed with a mixture of intermedine (80%) and lycopsamine (20%) produced danaidone at an average of 25.7 g per individual, which was comparable to that found in field-caught males. In contrast, a smaller amount of danaidone (5.7 to 7.0 g/male) was formed when males ingested retronecine or heliotrine, and those fed with an HCl salt of monocrotaline or retrorsine produced only traces of danaidone (< 0.5 g/male). In addition, males showed a strong feeding response to intermedine/lycopsamine, whereas the other PAs elicited no positive feeding behavior. These results indicate that, unlike the arctiid moths, P. sita males can only successfully convert limited chemical types of PAs into danaidone, and further suggest that in the field, males selectively ingest particular PAs that are readily transformable into danaidone.     

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.     

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.     

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).     

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.     

Protected by Fumigants: Beetle Perfumes in Antimicrobial Defense

Beetles share with other eukaryotes an innate immune system that mediates endogenous defense against pathogens. In addition, larvae of some taxa produce fluid exocrine secretions that contain antimicrobial compounds. In this paper, we provide evidence that larvae of the brassy willow leaf beetle Phratora vitellinae constitutively release volatile glandular secretions that combat pathogens in their microenvironment. We identified salicylaldehyde as the major component of their enveloping perfume cloud, which is emitted by furrow-shaped openings of larval glandular reservoirs and which inhibits in vitro the growth of the bacterial entomopathogen Bacillus thuringiensis. The suggested role of salicylaldehyde as a fumigant in exogenous antimicrobial defense was confirmed in vivo by its removal from glandular reservoirs. This resulted in an enhanced susceptibility of the larvae to infection with the fungal entomopathogens Beauveria bassiana and Metarhizium anisopliae. Consequently, we established the hypothesis that antimicrobial defense in beetles can be expanded beyond innate immunity to include external disinfection of their microenvironment, and we report for the first time the contribution of fumigants to antimicrobial defense in animals.     

Ethyl decanoate as a major component in the defensive secretion of two new zealand aleocharine (staphylinidae) beetles—Tramiathaea cornigera (broun) andThamiaraea fuscicornis (broun)

The defensive secretions of bothTramiathaea cornigera andThamiaraea fuscicornis contain undecane, toluquinone, ethyl decanoate and smaller amounts of other esters. The presence of esters particularly ethyl decanoate as a major component sets these two beetles apart chemically not only among the staphylinids but also within the subfamily Aleocharinae.     

Pyrrolizidine alkaloids in an overwintering population of monarch butterflies (Danaus plexippus) in California

California overwintering monarch butterflies contain both pyrrolizidine alkaloids (PAs) and theirN-oxides. Analysis of 76 individual monarchs by TLC, HPLC, GLC, and GC-MS has shown the presence of three types of PAs, the saturated diester sarracine, the saturated monoester 7-angelylplatynecine, and the unsaturated dialcohol retronecine. Monarchs arriving at the overwintering site in Santa Cruz, California, showed a wide variation in both the type and amount of PA present. Those sampled after a PA-containing plant (Senecio mikanioides) had bloomed at the site showed an altered PA profile. While the plant was found to contain sarracine and 7-angelylplatynecine, which are nontoxic to mammals, the monarchs showed an increase in retronecine levels, a toxic PA, after the plant bloom. Apparently monarchs utilize PA-containing plants both en route to their overwintering site and at the site, and potentially alter those PAs to forms toxic to mammals.     

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.     

Responses of two hymenopteran predators to surface Chemistry of their prey: Significance for an alkaloid-sequestering caterpillar

Larvae ofUresiphita reversalis (Guenée) (Lepidoptera: Pyralidae) sequester quinolizidine alkaloids from their leguminous hosts and store them primarily in the cuticle. Stored alkaloids are lost with the last larval molt. Extracts of late-instar larvae and of pupae were applied to larvae of the potato tuber moth,Phthorimaea operculella (Zeller) (Gelichiidae), which are normally palatable to two hymenopteran predators, the Argentine ant,Iridomyrmex humilis (Mayr) (Formicidae), and the paper wasp,Mischocyttarus flavitarsus (Sauss.) (Vespidae). Larvae ofP. operculella treated with alkaloid extracts ofU. reversalis larval exuviae, or with surface extracts of whole larvae, were deterrent to both predators, compared to untreated prey. Extracts of pupal exuviae added toP. operculella, however, were not deterrent.P. operculella larvae treated with the authentic alkaloids sparteine and cytisine were also deterrent to these hymenopteran predators. Storage of small but concentrated amounts of plant secondary compounds in the cuticle appears to be an efficacious means of defense against at least two common predators of lepidopteran larvae.     

Dabbing and shooting of benzo- and naphthoquinone secretions: Defensive strategies of bark-inhabiting aleocharine (col.: Staphylinidae) and tenebrionid (col.: Tenebrionidae) beetle larvae

The abdominal glands of three bark-inhabiting larvae of generaBolitochara, Leptusa (Staphylinidae), andHypophloeus (Tenebrionidae) were studied chemically and morphologically. Behavior of the larvae indicated that secretion is emitted only after severe disturbance of the larvae. These mechanical contacts may also occur incidentally with coinhabiting nonpredatory arthropods when the beetle larvae move within small interstices under bark. Depending on the species, the secretions contained 1,4-benzoquinone derivatives and three 6-alkyl-naphthoquinones dissolved within various alkanes, alkenes, ethyl-, isopropyl-, and isoamylesters. More erratically distributed gland constituents also detected were acetophenone, benzyl propionate, and methyl hydroxybenzoate. In the laboratory, synthetic quinone-containing solutions simulating those found inLeptusa andBolitochara larvae acted as strong topical irritants and caused further damage to last-stageCalliphora vomitoria larvae if hydrocarbons or esters were used as solvents. The natural secretions ofHypophloeus versipellis elicited considerable mortality in two subcortical sciarid larvae cooccurring with tenebrionid larvae. Bioassay and secretion chemistry of the Staphylinidae/Tenebrionidae larval secretions indicated that they are typical defensive secretions that act topically. Morphological data characterized Bolitocharini larvae as possessing protuberant abdominal tergites supplied with an interiorly situated gland reservoir. After mechanical contact, the defensive secretion is topically applied to other arthropods by dabbing this tergal protuberance on targets. The defensive gland ofHypophloeus versipellis is unusual in possessing a movable reservoir opening situated at the anterior border of tergite IX. By this peculiar gland morphologyHypophloeus larvae are capable of shooting secretion droplets frontally from their slightly depressed dorsal abdominal surface without bending their abdominal tips dorsally. This seems an adaptation to the interstitial habitat of the larvae. The types of defensive glands and their phylogenetic value in Aleocharinae/Tenebrionidae larvae are discussed.     

Scheloribatid Mites as the Source of Pumiliotoxins in Dendrobatid Frogs<Superscript>†</Superscript>

The strawberry poison frog Dendrobates pumilio (Anura: Dendrobatidae) and related poison frogs contain a variety of dendrobatid alkaloids that are considered to be sequestered through the consumption of alkaloid-containing arthropods microsympatrically distributed in the habitat. In addition to ants, beetles, and millipedes, we found that adults of two species of oribatid mites belonging to the cohort Brachypylina, trophically a lower level of animal than ants and beetles, contain dendrobatid alkaloids. Gas chromatography/mass spectrometry (GC/MS) of hexane extracts of adult Scheloribates azumaensis (Oribatida: Acari) revealed the presence of not only pumiliotoxin 251D (8-hydroxy-8-methyl-6-(2′-methylhexylidene)-1-azabicyclo[4.3.0]nonane), but also precoccinelline 193C and another coccinelline-type alkaloid. From the corresponding extracts of an unidentified Scheloribates sp., pumiliotoxin 237A (8-hydroxy-8-methyl-6-(2′-methylpentylidene)-1-azabicyclo[4.3.0]nonane) was detected as a minor component, and identified by synthesis. The presence of related alkaloids, namely deoxypumiliotoxin 193H, a 6,8-diethyl-5-propenylindolizidine, and tentatively, a 1-ethyl-4-pentenynylquinolizidine, were indicated by the GC/MS fragmentation patterns, along with at least another six unidentified alkaloid components. Thus, one possible origin of pumiliotoxins, coccinellid alkaloids, and certain izidines found in poison frogs may be mites of the genus Scheloribates and perhaps related genera in the suborder Oribatida. Chemical Ecology of Oribatid Mites IV.     

Composition of larval secretion ofChrysomela lapponica (Coleoptera,Chrysomelidae) and its dependence on host plant

The defensive secretion ofChrysomela lapponica larvae, which is produced by nine pairs of exocrine dorsal glands, has been chemically analyzed. TheC. lapponica larvae were kept in the laboratory on leaves of either birch (Betula pendula), alder (Alnus glutinosa), or willow (Salix fragilis). Larvae developed normally on birch and willow, whereas those on alder died within a few days. GC-MS analyses of the secretion of larvae on birch and willow revealed that the composition of this secretion differs distinctly from the known ones of several otherChrysomela species feeding exclusively on Salicaceae. In the exocrine secretion of larvae on birch, 69 compounds were identified, which included the main components isobutyric acid, 2-methylbutyric acid, and esters of the two. Several of the esters have not been reported previously from nature. The alcoholic components of the esters may be hydrolysis products ofBetula glycosides. Most components of the secretion of larvae feeding on birch were also found in the secretion of larvae feeding on willow. In addition, major amounts of benzoic acid and salicylalcohol were present in the secretion of the larvae feeding on willow.C. lapponica obviously acquires salicylalcohol by hydrolysis of salicin from willow leaves. However, in contrast to otherChrysomela species,C. lapponica larvae oxidize only traces of salicylalcohol to salicylaldehyde. The repellent activity of single authentic compounds of the secretion of larvae feeding on birch and willow, respectively, was tested in laboratory bioassays with ants (Myrmica sabuleti). Biosynthetic pathways to some identified compounds are suggested and discussed under evolutionary and functional aspects.     

Quantitative and qualitative effects of larval diet on male scent secretions ofEstigmene acrea,Phragmatobia foliginosa,andPyrrharctia isabella (Lepidoptera: Arctiidae)

In feeding experiments with insects reared in the laboratory, the presence of the dihydropyrrolizines hydroxydanaidal and danaidal in the male scent organs (coremata) of the arctiids,Estigmene acrea (Drury),Phragmatobia fuliginosa (L.), andPyrrharctia isabella (J.E. Smith), was shown to depend on the presence of a source of pyrrolizidine alkaloids (PAs) in the larval diet.Phragmatobia males given an artificial diet supplemented with the powdered roots of the PA-containing plantSymphytum officinale L. (comfrey) produced more hydroxydanaidal than danaidal, whereas males given an artificial diet supplemented with dried whole plants of another PA-containing species,Senecio vulgaris L., produced more danaidal than hydroxydanaidal.Pyrrharctia males produced hydroxydanaidal with little if any danaidal, whether the source of PAs was comfrey orS. vulgaris. A behavioral bioassay showed that the coremata of PA-deniedPyrrharctia male progeny of PA-denied parents were pheromonally inactive, whereas those of PA-denied male progeny of PA-supplied parents (male and/or female) were often active. This indicates that a small amount of pheromone is made from PAs transferred from the female to her eggs and that males effect copulatory transfers of PAs that are, in turn, passed to the eggs by the mated female. Field observations ofPhragmatobia andPyrrharctia larvae feeding on sources of PAs were reported. The PA monocrotaline was shown to be a feeding stimulant forPyrrharctia larvae.