Biosynthesis of the Sex Pheromone Component (E,Z)-7,9-Dodecadienyl Acetate in the European Grapevine Moth, Lobesia botrana, Involving ∆11 Desaturation and an Elusive ∆7 Desaturase

The European grapevine moth, Lobesia botrana, uses (E,Z)-7,9-dodecadienyl acetate as its major sex pheromone component. Through in vivo labeling experiments we demonstrated that the doubly unsaturated pheromone component is produced by ∆11 desaturation of tetradecanoic acid, followed by chain shortening of (Z)-11-tetradecenoic acid to (Z)-9-dodecenoic acid, and subsequently introduction of the second double bond by an unknown ∆7 desaturase, before final reduction and acetylation. By sequencing and analyzing the transcriptome of female pheromone glands of L. botrana, we obtained 41 candidate genes that may be involved in sex pheromone production, including the genes encoding 17 fatty acyl desaturases, 13 fatty acyl reductases, 1 fatty acid synthase, 3 acyl-CoA oxidases, 1 acetyl-CoA carboxylase, 4 fatty acid transport proteins and 2 acyl-CoA binding proteins. A functional assay of desaturase and acyl-CoA oxidase gene candidates in yeast and insect cell (Sf9) heterologous expression systems revealed that Lbo_PPTQ encodes a ∆11 desaturase producing (Z)-11-tetradecenoic acid from tetradecanoic acid. Further, Lbo_31670 and Lbo_49602 encode two acyl-CoA oxidases that may produce (Z)-9-dodecenoic acid by chain shortening (Z)-11-tetradecenoic acid. The gene encoding the enzyme introducing the E7 double bond into (Z)-9-dodecenoic acid remains elusive even though we assayed 17 candidate desaturases in the two heterologous systems. Supplementary Information The online version contains supplementary material available at 10.1007/s10886-021-01252-3.


Introduction
The European grapevine moth, Lobesia botrana, belongs to the family Tortricidae (Lepidoptera). It feeds on grapes, causing serious yield losses as well as increasing susceptibility to fungal infections (Ioriatti et al. 2011). It is among the most economically serious pests in vineyards in Europe, as well as in Chile, Argentina and California, where L. botrana was accidentally introduced (Gonzales 2010; Varela et al. 2010;Witzgall et al. 2010). The use of sex pheromone-based strategies for pest control is considered an environmentally safe management approach. Pheromone-mediated mating disruption of L. botrana is an effective technique for grape protection and is currently used on about 140,000 ha in the European wine-growing area in the European Union (Ioriatti et al. 2011).
Compared to other organisms in which fatty acyl desaturases are largely involved in normal cellular lipid metabolism, moth desaturases have evolved to perform specialized functions in the biosynthesis of sex pheromone components. Desaturases introduce double bonds in specific positions of fatty acids, and are responsible for much of pheromone diversity among different moth species. A wide range of desaturases has been characterized in various moths species, including: Δ5 desaturases that introduce double bonds into tetradecanoic acid for production of the fatty acid pheromone precursor (Z)-5-tetradecenoic acid in Ctenopseustis obliquana and C. herana (Hagström et al. 2014); a Δ6 desaturase that introduces an E6 double bond into the major pheromone component of Antheraea pernyi (Wang et al. 2010); several Δ9 desaturases (from a range of species) that introduce double bonds into saturated or unsaturated fatty acids of C 12 -C 18 chain length Liu et al. 2002;Rodríguez et al. 2004;Rosenfield et al. 2001); a Δ10 desaturase that introduces a double bond in hexadecanoic acid to produce the pheromone precursor (Z)-10-hexadecenenoic acid in Planotortrix octo ; several Δ11 desaturases that produce Δ11-unsaturated fatty acids (Knipple et al. 1998); two Δ11/Δ12 desaturases in Spodoptera exigua and S. litura that introduce double bonds into both saturated and unsaturated fatty acids to produce (Z)-11-hexadecenoic acid (Z11-16:acid) and (Z,E)-9,12-tetradecadienoic acid (Xia et al. 2019); a Δ11/Δ13 multifunctional desaturase in Thaumetopoea pityocampa that produces Z11-16:acid, 11hexadecynoic acid and (Z)-13-hexadecen-11-ynoic acid (Serra et al. 2007); Δ14 desaturases in Ostrinia species that introduce double bonds into palmitic acid to produce (Z)-and (E)-14-hexadecenoic acids ; and a terminal desaturase in Operophtera brumata that introduces a double bond into the methyl terminus of the carbon chain of Z 1 1 , Z 1 4 , Z 1 7 -e i c o s a t r i e n o i c a c i d t o p r o d u c e Z11,Z14,Z17,19-eicosatetraenoic acid (Ding et al. 2011).
In addition to desaturases, moth pheromone biosynthesis involves other enzymes that contribute to structural diversity. β-Oxidases and elongases are considered to combine with desaturases to determine the basic structures of pheromone fatty acyl precursors (Löfstedt et al. 2016) but, so far, no enzymes involved in chain-shortening havebeen identified and characterized. Fatty-acyl reductases (FARs) with different substrate specificities are responsible for reducing fatty acyl moieties to alcohols, and have been functionally characterized in several moth species (Lassance et al. 2010;Löfstedt et al. 2016;Moto et al. 2003).
In the present study, we performed in vivo labeling experiments to investigate the pheromone biosynthetic pathway in L. botrana. We did high-throughput sequencing of the L. botrana pheromone gland transcriptome and identified candidate genes that might be involved in pheromone biosynthesis. Finally, we functionally characterized several of these candidate genes in yeast and Sf9 heterologous systems.

Methods and Materials
Insects Pupae of L. botrana were obtained from a rearing facility at the Julius Kühn Institut (JKI), Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Fruit Crops and Viticulture, Siebeldingen, Germany. Larvae were reared in 500 ml polypropylene (PP) cups (Huthamaki, Alf, Germany) on a semi-synthetic diet, according to the protocol described by Markheiser et al. (2018). Male and female pupae were kept separately in a climate chamber at 23 ± 1°C under a 17 h:7 h Light: Dark photoperiod and 70% RH. After emergence, adults were fed with 10% honey solution, with two-to three-day-old virgin females being used for experiments throughout this study.

Labeling Experiments and Sample Preparation
The deuterium-labeled potential precursor acids D 3 -16:acid, D 3 -14:acid, D 3 -12:acid, D 5 -Z11-14:acid, and D 3 -Z9-12:acid were dissolved separately in dimethylsulphoxide (DMSO) at 40 μg/μl. About 1 h into scotophase, 0.4 μl of a solution of a potential precursor was applied topically to the pheromone gland of females in a group. The same volume of DMSO was applied as a control to females in another group. After 1 h incubation, pheromone glands were excised and five glands pooled in a 250 μl insert (in a 1.5 ml glass vial) to which 20 μl n-heptane was added. After extracting glands for 30 min at room temperature, the solvent, which contained pheromone components, was transferred into a new vial. The remaining lipids in the residue were subsequently extracted with 100 μl chloroform/methanol (2:1 v:v) at room temperature overnight. After extraction, base methanolysis was conducted as described in Bjostad and Roelofs (1984).

Gas Chromatography/Mass Spectrometry (GC/MS)
Pheromone gland extracts and methylated fatty acyl compounds were analyzed using a Hewlett-Packard (HP) 5975 mass selective detector coupled to an HP 6890 series gas chromatograph, equipped with a polar column (HP-INNOWax, 30 m × 0.25 mm, 0.25 μm film thickness) or an Agilent 5975C mass selective detector coupled to an Agilent 7890A series gas chromatograph equipped with a non-polar column (HP-5MS, 30 m × 0.25 mm, 0.25 μm film thickness). Helium was used as carrier gas (average velocity: 33 cm sec −1 ). The injector was configured in splitless mode at 250°C.
For analysis of pheromone gland extracts, the column oven temperature was set at 80°C for 1 min, then increased to 190°C at 10°C min −1 , held for 10 min, and finally increased to 230°C at 4°C min −1 , then held for 10 min. Incorporation of deuterium label into the pheromone components was detected by selected ion monitoring (SIM) mode (Table 1).
For fatty acid methyl esters (FAMEs), the oven temperature was set at 80°C for 1 min, then increased to 230°C at 10°C min −1 and held for 10 min. Incorporation of deuterium label into pheromone precursors was detected in the SIM mode, as described in Table 2. Full scans, from m/z 30-400, were for mass spectra. Compounds were identified by comparison of retention times and mass spectra with corresponding standards.

RNA Isolation, cDNA Library Construction and Illumina Sequencing
Approximately 50 pheromone glands of two-to three-day-old virgin females of L. botrana were collected for transcriptome sequencing. Total RNA of the glands was extracted using the TRIzol® reagent (Invitrogen). As control tissue, 25 abdominal tips from males were collected and treated the same way. We used Agilent 2100 Bioanalyzer system to check the RNA integrity and quantitation. Total RNA was sent to Novogene (Hong Kong) for Illumina sequencing.

De Novo Assembly and Bioinformatics Analysis
Transcriptome assembly was accomplished using Trinity (Grabherr et al. 2011) to assemble the clean reads de novo. Gene function was annotated based on the databases of Nr (NCBI non-redundant protein sequences), Nt (NCBI nonredundant nucleotide sequences), Pfam (Protein family), KOG/COG (Clusters of Orthologous Groups of proteins), Swiss-Prot (a manually annotated and reviewed protein sequence database), KO (KEGG Ortholog database) and GO (Gene Ontology).

Quantification of Gene Expression Level
Gene expression levels were estimated by RSEM (Li and Dewey 2011) for each sample: clean data were mapped back onto the assembled transcriptome and read count for each gene was obtained from the mapping results. Gene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) was implemented by the GOseq R packages based on Wallenius non-central hyper-geometric distribution (Young et al. 2010), which can adjust for gene length bias in DEGs. They were converted into values of FPKM (expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs-sequenced in RNA-seq). FPKM is the most common method of estimating gene expression levels, which considers the effects of both sequencing depth and gene length on counting of fragments (Van Verk et al. 2013).

Functional Assay in Yeast
For the construction of a yeast expression vector containing a candidate desaturase gene, specific primers with attB1 and attB2 sites incorporated were designed for amplifying the ORF. The PCR products were subjected to agarose gel electrophoresis and purified using the Wizard® SV Gel and PCR Clean up system (Promega). The ORF was cloned into the pDONR221 vector in the presence of BP clonase (Invitrogen). After confirmation by sequencing, the correct entry clones were selected and subcloned to pYEX-CHT vector (Patel et al. 2003), and recombinant constructs analyzed by sequencing. The pYEX-CHT recombinant expression vectors harboring the L. botrana desaturase genes were introduced into the double deficient ole1/elo1 strain (MATa elo1::HIS3 o l e 1 : : L E U 2 a d e 2 h i s 3 l e u 2 u r a 3 ) o f t h e y e a s t Saccharomyces cerevisiae, defective in both desaturase and elongase functions (Schneiter et al. 2000), using the S.c. easy yeast transformation kit (Life technologies). For selection of uracil and leucine prototrophs, the transformed yeast was allowed to grow on SC plate containing 0.7% YNB (w/o aa, with ammonium sulfate), a complete dropout medium lacking uracil and leucine (Formedium), 2% glucose, 1% tergitol (type Nonidet NP-40, Sigma), 0.01% adenine (Sigma) and 0.5 mM oleic acid (Sigma) as an extra fatty acid source. After 4 days at 30°C, individual colonies were picked up to inoculate 10 ml selective medium, which was grown at 30°C at 300 rpm for 48 h. Yeast cultures were diluted to an OD600 of 0.4 in 10 ml fresh selective medium containing 2 mM CuSO 4 with supplementation of a biosynthetic precursor. Each FAME precursor (14:Me, E9-14:Me, Z11-14:Me, Z9-12:Me) was prepared at a concentration of 100 mM in 96% ethanol and added to reach a final concentration of 0.5 mM in the culture medium (Ding and Löfstedt 2015;Ding et al. 2011). We used FAMEs as supplemented precursors (here and in the assay with insect cell lines below) because they are more soluble in the medium than free fatty acids. Yeasts were cultured at 30°C with Cu 2+ -induction. After 48 h, yeast cells were harvested by centrifugation at 3000 rpm and the medium was discarded. The pellets were stored at −80°C until fatty acid analysis.

Functional Assay in Sf9 Cells
The expression construct for Lbo_ACOs in the BEVS donor vector pDEST8 was made by LR reaction. Recombinant bacmids were made according to instructions for the Bac-to-Bac™ system given by the manufacturer Invitrogen using DH10MEmBacY (Geneva Biotech). Baculovirus generation was done using Sf9 cells (Invitrogen), Ex-Cell 420 medium (Sigma) and baculoFECTIN II (OET). Virus was then amplified once to generate a P2 virus stock using Sf9 cells and Ex-Cell 420 medium. The virus titer in the P2 stock was determined using the BaculoQUANT all-in-one qPCR kit (OET). Insect cells lines Sf9 were diluted to 2 × 10 6 cells/ml. Expression was done in 20 ml cultures in Ex-Cell 420 media, at virus additions (MOI 1). The cultures were incubated in 125 ml Erlenmeyer flasks (100 rpm, 27°C), with Z11-14:Me supplemented at a concentration of 0.25 mM on the second day. On the fourth day of expression, 7.5 ml samples were taken from the culture and centrifuged for 15 min at 4500 x g at 4°C. The pellets were stored at −80°C until fatty acid analysis. Aliquots were also taken for visualization in the fluorescence microscope of YFP expression from the virus backbone.

Fatty Acid Analysis of Yeast and Sf9 Cells
Total lipids were extracted using 3.75 ml of methanol/ chloroform (2:1, v/v) in a glass tube. One ml of HAc (0.15 M) and 1.25 ml of water were added to the tube to wash the chloroform phase. Tubes were vortexed vigorously and centrifuged at 2000 rpm for 2 min. The bottom chloroform phase (ca. 1 ml), containing the total lipids, was transferred to a new glass tube. FAMEs were made from this total lipid extract. The solvent was evaporated to dryness under gentle nitrogen flow. One milliliter of sulfuric acid, 2% in methanol, was added to the tube, vortexed vigorously, and incubated at 90°C for 1 h. After incubation, 1 ml of water was added, mixed well, and then 1 ml of n-hexane used to extract FAMEs. FAME samples were subjected to GC/MS analysis on an Agilent 8890 GC/Agilent 7693MS.

Transcriptome Assembly
A total of more than 78 million raw reads were generated by Illumina HiSeq™ 2500 from the pheromone glands of L. botrana, resulting in about 75 million clean reads after clustering and redundancy filtering of the raw reads. Data were deposited in NCBI database under accession code PRJNA663283. The clean reads were assembled into 75,207 unigenes with a mean length of 1247 bp and the N50 length of 2061 bp (Table 3). BUSCO completeness for the assembled transcripts was 96% (Simao et al. 2015).

Gene Ontology (GO) Annotation
The 75,207 unigenes were classified into different functional groups using BLAST2GO for Gene Ontology (GO) annotation. Based on sequence homology, 29,065 unigenes (38.64%) could be annotated. After GO annotation, the successfully annotated genes were grouped into three main GO domains: Biological Process (BP), Cellular Component (CC), Molecular Function (MF). One unigene could be annotated  Fig. 1 Fatty acid profile of Lobesia botrana female pheromone glands. Mass chromatograms of fatty acids in the Lobesia botrana pheromone gland in the form of fatty acid methyl esters. Acronyms refer to geometry across double bonds, position of unsaturation, carbon chain length and esterification; e.g., E9-12:Me refers to methyl (E)-9-dodecenoate into more than one GO term. Each unigene was grouped into one or more GO domains (Fig. 4).

Phylogenetic Analysis
By searching the transcriptome data using desaturase His 1, 3 family motifs and the cytochrome b5 domain (Marquardt et al. 2000;Napier et al. 1999), we found 17 full-length desaturaselike genes. We next performed phylogenetic reconstructions with the 17 desaturase-like genes identified in L. botrana. Five genes fall into the front-end/cytochrome-b5-related clade (Fig.  S2). These were subsequently treated separately due to their low similarity with first desaturases. Our analyses indicate that three of the L. botrana first desaturase candidates cluster into The deuterium-labeled compounds elute earlier than unlabeled compounds because of isotope effects (Matucha et al. 1991) the Δ9 desaturases clade, seven fall into the clade of Δ11, Δ10 and bifunctional desaturases, and the last two did not cluster into any functionally-characterized desaturase clade (Fig. 5).

Functional Assay of Desaturase Candidates
We heterologously expressed all the desaturase candidates in our Δole1/elo1 yeast system. In the first round of experiments, we fed the yeast with 14:Me as substrate; the yeast naturally produces a high amount of saturated C 16 fatty acid precursor. Lbo_KPSE, Lbo_NPVE and Lbo_GATD produced Z9-14:acid and Z9-16:acid. Lbo_PPTQ produced Z11-14:acid, Lbo_LPGQ produced Z11-16:acid and (Z)-11-octadecenoic acid (Z11-18:acid). Lbo_TPSQ showed Δ12 desaturation activity, producing (Z)-12-tetradecenoic acid (Z12-14:acid), (Z)-12-hexadecenoic acid (Z12-16:acid), and (Z)-12octadecenoic acid (Z12-18:acid). We assigned the name "group A" for these 6 desaturases (Fig. 6a). For those not showing any activity in this round, the name "group B" was assigned. In the second round of experiments, we fed the yeast expressing each desaturase candidate with Z9-12:Me, but none of the desaturases showed any evidence of Δ7 desaturation (Fig. 6b). The chromatogram is from Lbo_PPTQ, but is representative of all the desaturases from Group A and Group B. We further supplemented group B desaturases with E9-14:Me and Z11-14:Me, but did not see any doubly unsaturated product (E9,Z11-14:Me). We also expressed all the group B desaturases in the Sf9 system (Fig. 7). Thus, in the third round of experiments, all the "group B" desaturase were fed with 14:Me to test if there were any activity at all in the Sf9 cells, since they are not active in the yeast system. Figure 7c is a chromatogram from Lbo_SPTQ fed with 14:Me, which is representative of all the candidates from "group B". None of them showed any desaturation activity. In the fourth round of experiments, all "group B" desaturases and the Lbo_PPTQ were fed with Z9-12:Me in Sf9 cells. None of them showed Δ7 desaturation activity, neither producing any Δ7 unsaturated monoenes nor dienes. The chromatogram from Lbo_SPTQ fed with Z9-12:Me (Fig. 7d) is representative of all the candidates from "group B", with all resulting in similar chromatograms.
We found a significant peak of Z9-12:Me in the chromatograms of cells expressing ACO_31670 and ACO_49602 when Z11-14:Me was added, but only a tiny peak in cells harboring the empty virus (two replicates). Hypothesizing that Lbo_PPTQ introduces the first double bond in tetradecanoic acid and that the second double bond is introduced by another desaturase immediately after chain shortening of the Z11-14:acid intermediate, we also co-expressed Lbo_PPTQ, ACO_31670, ACO_49602 and all "group B" desaturases in Sf9 cells in a separate experiment. We did not find any trace of methyl E7,Z9-12:Me (data not shown).

Discussion
In the present study, we investigated the sex pheromone biosynthetic pathways in L. botrana. As shown in Fig. 9, our in vivo labeling experiment proved that the sex pheromone is biosynthesized from chain-shortening of 16:acid to 14:acid, followed by Δ11 desaturation to produce Z11-14:acid, which is further chain-shortened to Z9-12:acid. Subsequently, an unusual Δ7 desaturation occurs on Z9-12:acid to produce the precursor, E7,Z9-12:acid, which undergoes further reduction and acetylation to the corresponding alcohol and acetate. Three geometrid moths, Idaea aversata, I. straminata and I. biselata, use different 7,9-dodecadienyl acetates as pheromone components, with (Z,Z)-9,11-tetradecadienyl acetate acting synergistically in field trapping of I. aversata (Ando et al. 1987;Biwer et al. 1975;Szőcs et al. 1987;Zhu et al. 1996). When D3-16:acid was applied to pheromone glands of I. aversata, label was incorporated into both Z9,Z11-14:OAc and Z7,Z9-12:OAc suggesting that, in this case, the dodecadienyl precursor is a chain-shortening product of the longer C 14 homolog (Zhu et al. 1996). However, there is no evidence for a similar pathway in L. botrana because no (E,Z)-9,11-tetradecadienoic acid, neither native compound nor deuterium-labeled, was found in the pheromone gland after application of D 5 -Z11-14:acid. The incorporation of deuterium label from D 5 -Z11-14:acid into Z9-12:acid indicated chainshortening of the monounsaturated tetradecenyl precursor to produce the dodecenyl intermediate (Fig. 2a). Trace-level label incorporation was found from D 3 -16:acid and D 3 -14:acid into E7,Z9-12:acid but not into Z9-12:acid, possibly because the amount of labeled monounsaturated intermediate was below the detection limit, or the Δ7 desaturase was highly active converting all the Z9-12:acid.
The subsequent functional assays of candidate genes in Δole1/elo1 yeast and Sf9 expression systems demonstrated that Lbo_PPTQ is a Δ11 desaturase working on 14:acid to produce Z11-14:acid, which is consistent with the results of the labeling experiments. In addition, our functional assays of all the other desaturase gene candidates confirmed that Lbo_KPSE, Lbo_NPVE, and Lbo_GATD are Δ9 desaturases, as suggested by phylogenetic analysis, and Lbo_LPGQ is a Δ11 desaturase forming predominantly Z11-18:acid and Z11-16:acid from 18:acid and 16:acid, respectively. Lbo_TPSQ is a Δ12 desaturase working on 14:acid, 16:acid and 18:acid. Although these showed desaturase activity in the functional assays, there was no evidence that these five Δ9, Δ11 and Δ12 desaturases are   (Matoušková et al. 2007;Moto et al. 2004;Serra et al. 2006;Xia et al. 2019). In the codling moth, Cydia pomonella, (E,E)-8,10-dodecadienol (E8,E10-12:OH) is biosynthesized by a bifunctional E9 desaturase working on 12:acid (Löfstedt and Bengtsson 1988). The aliphatic carbon chain length in moth pheromone compounds is adjusted by limited β-oxidation (Jurenka et al. 1994), which is considered to be performed by four enzymes, an acyl-CoA oxidase, an enoyl-CoA hydratase, a L-3hydroxyacyl-CoA dehydrogenase, and a thiolase, as discussed in Ding and Löfstedt (2015), with several candidate genes suggested in Agrotis segetum in the same study. The first step of this β-oxidation is catalyzed by an acyl-CoA oxidase with different specificities (Osumi and Hashimoto 1978). As mentioned above, our labeling experiments demonstrated that, in the L. botrana pheromone gland, a β-oxidation enzyme was involved in producing the biosynthetic intermediate Z9-12:acid through chain shortening of Z11-14:acid. We found three full-length acyl-CoA oxidase (ACO) gene candidates from the transcriptome data, and heterologously expressed the two ACOs with the highest expression levels in the Sf9 system. The results showed that both Lbo_31670 and Lbo_49602 could chain-shorten Z11-14:acid to Z9-12:acid, but no shorter chain-length acids were found (Fig. 8).  Studies of pheromone biosynthetic pathways have demonstrated that chain-shortening is a crucial step in pheromone biosynthesis in many moth species (Bjostad and Roelofs 1983;Bjostad et al. 1987;Jurenka et al. 1994;Jurenka 1997;Xia et al. 2019;Wolf and Roelofs 1983;Wu et al. 1998). Differences in chain-shortening result in the production of different sex pheromone component ratios in two strains of the cabbage looper, Trichoplusia ni, i.e., the normal T. ni preferentially chain-shortened Z11-16:acid through two cycles of β-oxidation to Z7-12:acid, whereas mutant strain females had a reduced ability to chain-shorten (Jurenka et al. 1994). In the turnip moth, Agrotis segetum, successive β-oxidations starting from Z11-16:acid produced Z9-14:acid, Z7-12:acid, and Z5-10:acid. These three acids were then reduced and acetylated into the pheromone components Z9-14:OAc, Z7-12:OAc and Z5-10:OAc (Löfstedt et al. 1986). Differences in chain-shortening activity account for different ratios of these pheromone components in Swedish (12:59:29) and Zimbabwean (78:20:2) populations of A. segetum (Wu et al. 1998).
Identification of the genes encoding limited β-oxidation enzymes should help us understand the molecular control of chain shortening. The most important step here is the first, with the acyl-CoA oxidase catalyzing the formation of a double bond between the second and third carbon. However, this functionality had not previously been characterized in any moth species in the context of pheromone biosynthesis. This is the first study to report functional ACO genes involved in pheromone biosynthesis. We suggest that the subsequent three reactions are performed by the Sf9 cell machinery and most likely by a protein with three functions and less specific to substrate chain length (Hashimoto 1996).
To date, no acetyltransferase gene has been characterized in the context of moth sex pheromone biosynthesis. By homology, searching in L. botrana, we failed to find any novel candidate genes to test for this activity other than the ones from A. segetum that had previously been tested with negative results (Ding and Löfstedt 2015).
In conclusion, we reveal the biosynthetic pathway for the pheromone of the European grapevine moth, L. botrana, including evidence that an unusual Δ7 desaturation activity is involved. We found six functional desaturase genes, of which Lbo_PPTQ exhibits high Δ11 desaturase activity on