Insect Biochemistry and Molecular Biology 41 (2011) 715e722 Contents lists available at ScienceDirect Insect Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/ibmb Terminal fatty-acyl-CoA desaturase involved in sex pheromone biosynthesis in the winter moth (Operophtera brumata) Bao-Jian Ding a,*, Marjorie A. Liénard a, Hong-Lei Wang a, Cheng-Hua Zhao b, Christer Löfstedt a a Functional Zoology, Department of Biology, Lund University, Sölvegatan 37, SE-223 62 Lund, Sweden b State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, The Chinese Academy of Sciences, 100080 Beijing, China article info abstract Article history: The winter moth (Operophtera brumata L., Lepidoptera: Geometridae) utilizes a single hydrocarbon, Received 1 April 2011 1,Z3,Z6,Z9-nonadecatetraene, as its sex pheromone. We tested the hypothesis that a fatty acid precursor, Received in revised form Z11,Z14,Z17,19-nonadecanoic acid, is biosynthesized from a-linolenic acid, through chain elongation by 16 May 2011 one 2-carbon unit, and subsequent methyl-terminus desaturation. Our results show that labeled a- Accepted 16 May 2011 linolenic acid is indeed incorporated into the pheromone component in vivo. A fatty-acyl-CoA desaturase gene that we found to be expressed in the abdominal epidermal tissue, the presumed site of biosynthesis Keywords: for type II pheromones, was characterized and expressed heterologously in a yeast system. The trans- Methyl-terminus desaturase In vivo labeling genic yeast expressing this insect derived gene could convert Z11,Z14,Z17-eicosatrienoic acid into Yeast expression Z11,Z14,Z17,19-eicosatetraenoic acid. These results provide evidence that a terminal desaturation step is Epidermal tissue involved in the winter moth pheromone biosynthesis, prior to the decarboxylation. Δ11-Desaturase Ó 2011 Elsevier Ltd. All rights reserved. Oenocyte cells Linolenic acid Hydrocarbon 1. Introduction the gypsy moth (Lymantria dispar L., Lymantridae), biosynthesis of the alkene precursor of its epoxide sex pheromone occurs in The sex pheromone of the winter moth Operophtera brumata oenocyte cells. The alkene is subsequently transported to the (Lepidoptera, Geometridae) was identified as 1,Z3,Z6,Z9- pheromone gland and converted into an epoxide (Jurenka et al., nonadecatetraene (1,Z3,Z6,Z9-19:H) (Bestmann et al., 1982; Roelofs 2003). In these species, the role of the pheromone biosynthesis et al., 1982). This unusual hydrocarbon belongs to and possesses activating neuropeptide (PBAN) is postulated to regulate the the common features of so-called type II pheromones of Lepidoptera precursor uptake into the pheromone gland, rather than regulation (Millar, 2000). Type II pheromones are polyene hydrocarbons and of a biosynthetic step or the epoxidation or release of pheromone related epoxides (and ketones in rare cases) with an odd number of (Wei et al., 2004; Ando et al., 2008). This is in contrast with the carbons (C17eC25), with double bonds occurring at positions 3,6,9, biosynthesis of type I pheromones, which is reported to be under or 6,9 with Z-configuration (Millar, 2000). They are biosynthetically direct PBAN control in many species (Raina et al., 1989; Rafaeli and derived from a-linolenic acid and/or linoleic acid (Millar, 2000) Jurenka, 2003; Rafaeli, 2009). through one or two cycles of 2-carbon unit chain elongation, and the Besides chain elongation of the a-linolenic acid and the final carboxyl group is removed by decarboxylation or decarbonylation decarboxylation reaction to convert the fatty acid to the hydro- (Rule and Roelofs, 1989; Ando et al., 2004; Jurenka, 2004). carbon, we postulated that the winter moth pheromone biosyn- Type II pheromone components that consist of hydrocarbons are thesis includes a methyl-terminus desaturation, which would reported to be produced in oenocyte cells and transported to the introduce a terminal double bond at the methyl-end of the fatty gland for release. However, if the pheromone blend comprises acid molecule. Desaturation of fatty acids is a common feature of epoxides, the final epoxidation occurs in the pheromone gland sex pheromone biosynthetic pathways in the Lepidoptera. The gene (Schal et al., 1998a; Wei et al., 2003, 2004; Matsuoka et al., 2006). In family of fatty-acyl-CoA desaturases includes members encoding enzymes displaying different substrate-, stereo- and regio- specificities (Knipple et al., 2002; Roelofs and Rooney, 2003). Two “Δ ” “Δ > ” * Corresponding author. Tel.: þ46 46 222 0126; fax: þ46 46 222 4716. desaturase subfamilies, the so-called 11 and 918C 16C E-mail address: [email protected] (B.-J. Ding). appear to be Lepidoptera-specific and can account for most of the 0965-1748/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2011.05.003 716 B.-J. Ding et al. / Insect Biochemistry and Molecular Biology 41 (2011) 715e722 pheromone structure variation among moth species (see for was examined by amplifying a short fragment of both the b-actin instance Liénard et al., 2008 and references therein). The Δ11 clade gene and the 16S rRNA gene. contains not only enzymes with Δ11 functionality but also Δ10, Δ11/ Δ10,12 and Δ11/Δ11,13 activities (Hao et al., 2002; Moto et al., 2004; 2.4. Characterization of desaturase-encoding cDNAs Serra et al., 2006, 2007). In the current study, we investigated pheromone biosynthesis in PCR amplification was performed using cDNA from the the winter moth by means of in vivo labeling and by heterologous abdominal epidermal tissue as template with a pair of degenerate expression of a candidate desaturase gene in yeast. We report on primers (Px117: 50-ACN GCN GGN GCN CAY MGN YTN TGG-30, the characterization of a new member of the Lepidoptera-specific Px118: 50-TGR TGR TAR TTR TGR AAN CCY TCN CC-30) designed Δ11-desaturase gene family, encoding a desaturase that intro- based on the conserved TAGAHR and GEGFH histidine-rich motifs duces a double bond in the methyl-end of the fatty acid molecule. of desaturases (Rosenfield et al., 2001), on a Veriti Thermo Cycler using AmpliTaq Gold polymerase (Applied Biosystems, Stockholm, 2. Materials and methods Sweden). Cycling parameters were as follows: an initial denaturing step at 94 C for 5 min, 35 cycles at 94 C for 15 s, 55 C for 30 s, fi 2.1. Insect and tissue collection 72 C for 2 min followed by a nal extension step at 72 C for 10 min. Specific PCR products were ligated into a TOPO TA pCR 2.1 Adult wingless winter moth females were obtained from Gabor vector (Invitrogen). The ligation product was transformed into Szöcs (Department of Zoology, Plant Protection Institute, Hungarian Escherichia coli strain Top10 (Invitrogen). Plasmid DNAs were iso- Academy of Science, Budapest, Hungary), or were collected from lated according to standard protocols and recombinant plasmids oak trees (Quercus ssp. L.) (554205500N, 131200200E) in the vicinity were subjected to sequencing using universal M13 primers and the of Lund, in November (at night, 4 p.m. to 7 p.m.). The collected BigDye terminator cycle sequencing kit v1.1 (Applied Biosystems). wingless female adults were kept under outdoor conditions and the Sequencing products were EDTA/ethanol-precipitated, dissolved in females that displayed the calling behavior were regarded as formamide and loaded for analysis on a capillary 3130xl Genetic unmated individuals and used in the experiments. Different body analyzer (Applied Biosystems). Curated consensus sequences were parts were dissected from virgin females and stored separately used for BLAST searches and revealed the presence of 3 distinct Δ at À80 C until RNA extraction. cDNA transcripts including two ubiquitous 9-desaturase cDNAs. Rapid amplification of 30- and 50 cDNA termini was conducted for the last transcript using the BD biosciences RACE kit (Clontech, In 2.2. In vivo labeling vitro Sweden AB, Stockholm) and gene-specific primers (Table 1). In a order to verify its integrity and sequence information, the open The deuterium labeled -linolenic acid (9,10,13,13,15,16-D6- reading frame (ORF) was amplified using a pair of primers (Table 1) Z9,Z12,Z15-octadecatrienoic acid) was diluted in DMSO, at designed spanning before the start codon and after the stop codon. a concentration of 40 mg/mL, and 1mL was injected in the female m All partial and full-length desaturase cDNA sequences have been abdomen, using a 10- L-Hamilton syringe. After 24-h incubation, deposited in the GenBank database under the accession numbers the pheromone gland was extracted to monitor label incorporation HQ917684-HQ917686. into the pheromone component. Females injected with a DMSO- only solution were used as controls. 2.5. Sequence and phylogenetic analyses GCeMS analyses were performed on a HewlettePackard model e 5972 MSD in EI mode and interfaced with a Hewlett Packard 5890 Comparison of the newly isolated desaturase sequences to series II gas chromatograph equipped with a HP-INNOWax capillary sequence information available publicly was performed using the   m column (30 m 0.25 mm 0.25 m, Agilent technologies), and non-redundant database of Blastx search (Altschul et al., 1997). helium was used as carrier gas (20 cm/cm). The oven temperature Transmembrane regions were predicted in the TMHMM server 2.0 was set to 80 C for 1 min, then increased at a rate of 10 C/min up (Erik et al., 1998). Prediction of the endoplasmic retention signal to 210 C, followed by a hold at 210 C for 15 min, and then was performed using PSORT (http://psort.hgc.jp/form2.html). increased at a rate of 10 C/min up to 230 C followed by a hold at Desaturase sequences used for phylogenetic reconstructions were 230 C for 20 min. retrieved from the GenBank (http://www.ncbi.nlm.nih.gov) and the To detect incorporation of deuterium atoms from the D6-lino- SilkDB (http://silkworm.genomics.org.cn/) databases.