Insect Biochem. Vol. 20, No. 8, pp. 809-819, 1990 0020-1790/90 $3.00+ 0.00 Printed in Great Britain Pergamon Press pie LONG-CHAIN AND VERY LONG-CHAIN METHYL-BRANCHED ALCOHOLS AND THEIR ACETATE ESTERS IN PUPAE OF THE TOBACCO HORNWORM, MANDUCA SEXTA * DENNIS R. NELSON 1, CHARLOTTEL. FATLAND t, JAMESS. BUCKNER 1, MERTXE DE RENOBALES2'~ and GARY J. BLOMQUIST 2 tBiosciences Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, State University Station, Fargo, ND 58105 and :Department of Biochemistry, University of Nevada, Reno, NV 89557, U.S.A. (Received 16 April 1990; revised and accepted 1 August 1990) Abstract--Four homologous series of very long-chain methyl-branched alcohols (VLMA, C3s to >C44) were found in the internal lipids of developing male pupae of the tobacco hornworm, Manduca sexta, both as free alcohols and as acetate esters. The four major homologous series, with carbon chain backbones of 36-44 carbon atoms, consisted of a monomethyl, two dimethyls and a trimethyl-branched alcohol series. The major alcohol of each homologous series (with the corresponding alkane obtained by reduction in parentheses) was identified as 24-methyltetracontan-1-ol (17-methyltetracontane), 24,28-dimethyltetracon- tan-l-ol (13,17-dimethyltetracontane), 22,34-dimethyloctatriacontan-l-ol (5,17-dimethyloctatriacontane) and 22,26,34-trimethyloctatriacontan-l-ol (5,13,17-trimethyloctatriacontane). The minor components of the VLMA had backbones with an odd number of carbon atoms (37, 39, 41 and 43). Methyl branches of the minor components were identified on the 18- and 14,18-positions when numbered from the alkyl end of the chain. Also identified were minor amounts of long-chain methyl-branched alcohols (LMA, Css to C32). The major components in the "wax ester" TLC fraction were acetate esters of the LMA and VLMA. Key Word Index: insects, Lepidoptera, pupae, lipids, fatty alcohols, methyl-branched alcohols, acetate esters INTRODUCTION Novel very long-chain methyl-branched alcohols (VLMA, C36 to >C44):[: were recently identified among the internal lipids of mixed-sex pupae of the cabbage looper, Trichoplusia ni (Nelson et al., 1989). These alcohols and their esters (de Renobales et al., 1989), and long-chain methyl-branched hydrocar- bons (de Renobales et al., 1988) were the major lipid *A preliminary report was given at the American Society for Biochemistry and Molecular Biology Meeting, 1-5 May 1988 (Nelson and de Renobales FASEB J. 2, A1790, 1988) and 3-7 June 1990 (Nelson and Blomquist, FASEB J. 4, A1827, 1990). tPresent address: Inasmet, Barrio Igara s/n, E-20009 San Sebastian, Spain. ~The subscript for carbon atoms refers to the total number of carbon atoms in the molecule. Other abbreviations used are: TLC, thin-layer chromatography; CGC-MS, capillary gas chromatography-mass spectrometry; C/M, chloroform/methanol; H/E, hexane/diethyl ether; H/E/F, hexane/diethyl ether/formic acid; TMS, trimethyl silyl; LiAIH(, lithium aluminum hydride; BSA, N,O-bis(trimethylsilyl)acetamide; FFA, free fatty acid; FAME, fatty acid methyl ester; SFA, saturated fatty acid; UFA, unsaturated fatty acid; UFAME, unsatu- rated fatty acid methyl ester; ROH, alcohols; WE, wax ester; LMA, long-chain methyl-branched alcohols (C2~ to C52); VLMA, very long-chain methyl-branched alcohols (C3s to > C~); DAP, days after pupation. components synthesized from [l-~4C]acetate and [1-t4C]propionate during the pupal stage. The very long-chain methyl-branched alcohols were not found in the larval or adult stages of the cabbage looper nor in the surface lipids of the pupae. The methyl-branch positions of these aiochols were not the same as the methyl-branch positions of the major methylalkanes found in the surface wax and in the internal lipids. To determine if the same or similar very long-chain methyl-branched alcohols were components of the pupal stage of other insects, we examined developing male pupae of the tobacco hornworm, Manduca sexta. In this report, we achieve the following: estab- lish that the structures of the VLMA found internally in developing male pupae of the tobacco hornworm are identical to those found in developing pupae of T. ni; extend the approach to interpretation of the mass spectra of VLMA; identify a number of long-chain mono- and di-methyl-branched alcohols (LMA; Css to C32) not found in T. ni; and establish that the "wax ester" fraction contains the acetate esters of the LMA and VLMA. METHODS AND MATERIALS Insects Larvae of the tobacco hornworm, Manduca sexta, were reared (Bell and Joachim, 1976) on a high wheat germ diet (Reinecke et al., 1980). Insects were reared under long-day 809 IB 20/8---C