Adalinine (2), a new piperidine alkaloid, has been isolated from the European two-spotted ladybird beetle Adalia bipunctata, and its structure has been determined by spectroscopic methods. This alkaloid was also shown to be present in all the life cycle stages of Adalia bipunctata as well as in the adults of a related species, A. decempunctata. Many ladybird beetles (Coleoptera; Coccinellidae) exhibit a defense mechanism known as reflex bleeding. When disturbed or molested, they emit droplets of an yellowish fluid at the joints of their legs. The presence of defensive alkaloids in this fluid is well documented. Alkaloids belonging to many different structural families such as pyrrolidines, piperidines, homotropanes, 2-methylperhydro-9b-azaphenalenes, azamacrolides, "dimeric" alkaloids, and long-chain and quinoline derivatives have been characterized from these insects. The European two-spotted ladybird Adalia bipunctata L. has been found to contain the homotropane alkaloid adaline (1). The deterrent and toxic properties of 1 have been investigated. We recently decided to reexamine the chemical defense secretion of A. bipunctata, in the context of a research project aimed at understanding the relationships between different species of predators competing for the same aphid population. Analysis of a CHCl3-soluble extract of A. bipunctata adults by GC-MS showed the presence, in addition to adaline (1), of a minor, nitrogen-containing compound, amounting to about 10% of the concentration level of 1. We report here the isolation and structure determination of this new alkaloid 2, for which the name adalinine has been coined. The HREIMS of adalinine (2) showed its molecular formula to be C13H23NO2, thus requiring three degrees of unsaturation. The most intense fragment ions were observed at m/z 168.1383 (C10H18NO, [M+ - C3H5O]), 154.0873 (C8H12NO2, [M+ - C5H11]), and 112.076 (C6H10NO, [M+ - C5H11 - C2H2O]), suggesting the presence of a pentyl chain and a -CH2COCH3 moiety. The 13C-NMR spectrum displayed 13 carbon atom signals (two CdO, one C, eight CH2, and two CH3). One of the CdO groups was assigned to a ketone (13C-NMR δ 207.2; IR, νCO 1709 cm-1) and the other to an amide (13C-NMR δ 171.4; IR, νCO 1659 cm-1). The structure of adalinine was established by a 2D NMR study (1H-1H COSY, HMQC, HMBC) and is depicted as 2. The complete assignments of the 1H- and 13C-NMR spectra of 2 are reported in the Experimental Section. The absolute configuration of 2 was not determined. Both adaline (1) and adalinine (2) have been detected in adults, eggs and the first- to fourth-instar larvae of A. bipunctata. A GC-MS study of Adalia decempunctata adult extracts showed the presence of both adaline (1), already reported in this species by Pasteels et al., 2 and adalinine (2). The detection of 2 by GC and TLC in fresh ladybird extracts indicates that this compound is not an artifact of the isolation procedure. Despite being a piperidone derivative, 2 is obviously structurally related to the major alkaloid adaline (1). As the latter is a β-amino ketone, it could undergo a retro-Mannich reaction to afford the imine 3. Addition of H2O followed by oxidation would afford 2 (Scheme 1). However, because the biosynthetic pathway leading to 1 is as yet unknown, the precise relationship existing between the two compounds is still a matter of speculation. The study of adalinine (2) biosynthesis is currently underway in our laboratories.