Figure 1.
Schematic outlining the likely biosynthetic routes of the sex pheromone components of C. obliquana, C. herana, P. octo, and P. excessana.
Desat1, desat5 and desat6 correspond to the desaturase genes encoding a Δ9 desaturase with a preference for 16>18 carbon fatty acids, a Δ10-desaturase and a Δ9-desaturase with a preference for 18>16 carbon fatty acids, respectively. Desat? refers to a yet to be identified Δ5-desaturase. Chain shortening by β-oxidation is indicated by ‘−2C’. The minor products of the two Δ9-desaturases in P. excessana (desat1 and desat6) are indicated in brackets. We also note that Z10-14:OAc is a very minor (2%) component of the pheromone blend of P. octo (not shown).
Table 1.
Summary statistics for desaturases from Ctenopseustis and Planotortrix species.
Figure 2.
Phylogeny of 86 lepidopteran desaturases including those encoded by desat1-6 from Ctenopseustis and Planotortrix.
The phylogeny was constructed from protein sequences using PHYML implemented within Geneious using JTT distances. Complete amino acid sequence information was obtained from GenBank, along with desaturases predicted from the genomic sequence of Bombyx mori from the Silkmoth database. Sequences are abbreviated as following: Ape, Antherea pernyi; Ase, Ascotis selenaria; Ave, Argyrotaenia velutinana; Bmo, Bombyx mori; Cpa, Choristoneura parallela; Cro, Choristoneura rosaceana; Che, Ctenopseustis herana; Cob, Ctenopseustis obliquana; Cpo, Cydia pomonella; Epo, Epiphyas postvittana; Has, Helicoverpa assulta; Hze, Helicoverpa zea; Lca, Lampronia capitella; Mbr, Mamestra brassicae; Mse, Manduca sexta; Pex, Planotortrix excessana; Poc, Planotortrix octo; Pno, Planotortrix notophaea; Onu, Ostrinia nubilalis; Ofu, Ostrinia furnacalis; Osc, Ostrinia scapulalis: Obr, Operophtera brumata; Sli, Spodoptera littoralis; Tni, Trichoplusia ni; Tpi, Thaumetopoea pityocampa; Ypa: Yponomeuta padellus. After the abbreviated species name are the desaturase activity if known with NF = non-functional in pheromone biosynthesis; TerDesat = terminal desaturase activity; Z or E, geometry of the double bond. The GenBank accession numbers are given in brackets for previously described desaturases. Bootstrap values in percentages from 1000 bootstrap replicates supporting the three major clades (Δ9-desaturase 16C>18C, Δ9-desaturase 18C>16C, and Δ11-desaturase) and the groups containing the Ctenopseustis and Planotortrix desaturases, indicated by the red outline boxes, are given above the relevant branches.
Table 2.
Likelihood ratio tests between nested site-specific models.a
Table 3.
Putative positively selected sites and posterior probabilities under M8.
Figure 3.
GC-MS analyses of DMDS derivatives from methanolysed Cu2+-induced ole1 elo1 S. cerevisiae yeast supplemented with Z9-18:Me and transformed with (A and C) control pYEX-CHT vector, (B) pYEX-CHT-Pex-desat3 and (D) pYEX-CHT-Che-desat4. The chromatogram traces represent the ion currents obtained by selection of the characteristic ion of terminal and Δ6-DMDS adducts at m/z 61 (A and B) and m/z 175 (C and D), respectively. (E) and (F) represent the mass spectra for terminal C16 DMDS adducts (Δ15-16) (m/z 362 [M+], 61, 301 (A+) and 269 (A+-32)) and Δ6-16 DMDS adducts (m/z 362 [M+], 187, 175 (A+) and 143 (A+-32), respectively. The mass spectra for other DMDS adducts present in the extracts are not shown and displayed a spectrum with the characteristic ions at m/z 334 [M+], 61 and 273 for Δ13-14, at m/z 390 [M+], 61 and 329 for Δ17-18 and at m/z 334 [M+], 175 and 159 for Δ6-14, respectively.
Figure 4.
GC-MS analysis of methanolysed total lipid extracts from ole1 S. cerevisiae yeast transformed with YEpOLEX-Cob-desat6. (A) Total ion current (TIC) chromatogram of fatty acid methyl esters of yeast expressing the Cob-desat6 gene. (B) DMDS derivatives of methanolyzed YEpOLEX-Cob-desat6 yeast extracts. The chromatogram traces represent the ion current obtained by selection of the characteristic ion of Δ9-adducts at m/z 217.
Figure 5.
Gene expression of desat1, desat5, and desat6 in the pheromone gland and abdomen of virgin females in Ctenopseustis obliquana, C. herana, P. excessana, and P. octo relative to housekeeper genes.
In panel (A) the normalised expression levels in the pheromone gland [PG] of C. herana (Che; light brown; n = 20) and C. obliquana (Cob; dark brown; n = 21) was compared with those in the abdomen [Ab] of C. herana (n = 20) and C. obliquana (n = 21), while in panel (B) the normalised expression levels in the pheromone gland of P. excessana (Pex; light green; n = 39) and P. octo (Poc; dark green; n = 24) are compared with those in the abdomen of P. excessana (n = 39) and P. octo (n = 25). Bars are the mean normalized gene expression, with error bars representing SEMs. Different small case letters indicate significant differences between tissues and/or species at the 95% level using the Bonferroni correction for each desaturase gene.