Azamacrolides: a family of alkaloids from the pupal defensive secretion of a ladybird beetle (Epilachna varivestis).

Abstract

Defensive droplets from glandular hairs of the pupa of the Mexican bean beetle, Epilachna varivestis, contain a group of structurally novel alkaloids, the azamacrolides. The major constituent of this secretion, epilachnene, is shown to be (5Z)-11-propyl-12-azacyclotetradec-5-en-14-olide. The secretion also contains an epilachnadiene and trace amounts of three closely related components. (Fig. 1). Suspecting this fluid to be defensive, we exposed pupae to ant attacks in a series of laboratory presentations. The pupae were individually placed on the bottom of small Petri dishes that served as the foraging arenas for laboratory colonies of the ants (Leptothorax longispinosus). The results, which were videotaped, were consistent and dramatic. The moment an individual ant, in an exploratory approach to the pupa, contacted some of the glandular hairs, it backed away and cleansed itself. While retreating, it repeatedly dragged its mouthparts and/or antennae against the substrate. None of the pupae (n = 10; each in a different arena) were injured during the 10-min presentations, although each was contacted by dozens of ants during this period (all pupae eventually gave rise to adults). We proceeded to collect the secretory droplets for analysis and here report on the chemicals isolated from the fluid (5). MATERIALS AND METHODSSample Collection. Secretion from the glandular hairs, collected with microcapillaries, was sealed directly in glass tubes (1.8 x 20 mm) or extracted with ether or dichloromethane (100 ul). For NMR spectroscopy, a sample (500 pupae) was taken up in 400 ul of C2HC13 (2H, 99.96%).Analytical Procedures. Samples were analyzed by gas chromatography (GC) on a Hewlett-Packard (HP) 5890 instruThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. ment equipped with a split/splitless injector, a flame ionization detector, and an HP 3396A integrator. Undiluted samples in glass capillaries were injected by a solid sampling technique (6, 7); solutions were introduced by splitless injection. Low-resolution mass spectrometry (MS) was carried out with an HP 5890 gas chromatograph linked to a Finnigan ion-trap detector (ITD 800) or an HP mass selective detector. Chemical ionization mass spectra were obtained on the ITD with methane as the reagent gas. High-resolution mass spectra were obtained on a Kratos 890 instrument. Gas-phase IR spectra were obtained with an HP 5890 gas chromatograph linked to an HP 5965A IR detector. Unless otherwise noted, 1H and 13C NMR spectra were obtained in C2HC13 on a Varian XL-400 instrument.Derivatization Techniques. (i) Microhydrogenation. A small sample of secretion in ether (50 ,ul) was placed in a glass vial and =0.5 mg of 10%o Pd on activated charcoal was added. A balloon filled with hydrogen was attached to the vial. After 10 hr, 25 ul of ether was added and the supernatant was withdrawn and examined by GC/MS.(ii) A...

Mayolenes: Labile defensive lipids from the glandular hairs of a caterpillar ( Pieris rapae )

“…They are of less frequent occurrence in insects, but the chemistry of the secretion of insectan hairs, to the extent that it is known, can be of interest. Thus, for instance, the pupal hairs of certain coccinellid beetles have been shown to produce, in one species, a series of azamacrolides, and in others a combinatorial mixture of macrocyclic polyamines (15)(16)(17). The present discovery of yet another novel group of secondary products from the glandular hairs of an insect suggests that the study of such defensive materials can be rewarding.…”

Polyazamacrolides from ladybird beetles: Ring-size selective oligomerization

“…Higher oligomers with more than three of the units 9 and 10 as well as the cyclic ''monomers'' epilachnene (1) and epilachnadiene (2; ref. 4) were not detected in the secretion either by NMR or by GC-MS and HPLC-MS analyses.…”

“…3), forming macrocycles with well over 200 members (7). Although the biosynthesis of the PAMLs (3)(4)(5) has not yet been investigated, the PAMLs clearly seem to be related biosynthetically to the azamacrolides (1, 2) from E. varivestis.…”

Interactions among attending ants, brown-citrus aphids, and ladybugs in plants of Citrus sinensis

“…As a chemical defense, ladybeetles bleed (reflex bleeding), thus extravasating a tibial-femoral linking fluid in the adults and on the dorsal surface of larvae and pupae, with properties the ants dislike (Attygalle et al, 1993;Sloggett, 1998;Majerus et al, 2007). In various species of ladybugs, the defensive chemical components are synthesized by the individuals themselves (Jones and Blum, 1983).…”