About 160,000 lepidopteran species are known world wide, and each species is believed to establish a species-specific mating communication system for species conservation. Not only nocturnal moths but also diurnal moths utilize special sex pheromones as a chemical cue. Lepidopteran sex pheromones have been identified from more than 600 moth species and about three-quarters of them are composed of C₁₀–C₁₈ unsaturated fatty alcohols, their acetates, and aldehyde derivatives, including one, two, or three C=C bonds (Type I pheromones; Ando et al., 2004; Ando, 2011; El-Sayed, 2011). These components are biosynthesized from saturated fatty acyl intermediates, into which double bonds are introduced at definite positions by desaturases working in a pheromone gland (Jurenka, 2004). The double bonds locate at various positions, i.e., from the terminal methyl group to the next carbon to the functional group. In addition to the different chain lengths and kinds of terminal functional groups, the chemical structural varieties are attributed to the different positions of the double bonds (Ando et al., 2004).

Plusiinae is a well-assembled subfamily in Noctuidae, which is the biggest family in Lepidoptera, and includes many pest species secreting Type I pheromones. Since a pioneering study on the chemical communication of the cabbage looper, Trichoplusia ni (Berger, 1966), sex pheromones have been identified from 22 Plusiinae species (Ando, 2011; El-Sayed, 2011). Most of them secrete (Z)-7-dodecenyl acetate (Z7-12:OAc) as a main pheromone component and blend some structurally related compounds to make species-specific pheromones, suggesting that a primitive species in Plusiinae utilized Z7-12:OAc and speciated to many existing species while establishing the ability to produce new pheromone components. Z7-12:OAc is biosynthesized via Δ11-desaturation of a saturated C₁₆ acyl compound (16:Acyl), probably a CoA-derivative of palmitic acid (16:Acid; Bjostad and Roelofs, 1983), and this key step has been confirmed by identification of the gene encoding Δ11-desaturase from T. ni (Knipple et al., 1998). The Δ11-desaturation is an important biosynthetic step because β-oxidation of the produced (Z)-11-hexadecenyl intermediate in different degrees yields a series of monoenyl compounds unsaturated at the ω5-position, such as (Z)-9-tetradecenyl acetate (Z9-14:OAc) and (Z)-5-decenyl acetate (Z5-10:OAc). Among the Plusiinae species, Z9-14:OAc is secreted by Macdunnoughia confusa, and Z5-10:OAc is secreted by Anadevidia peponis as a minor pheromone component (Inomata et al., 2000).

On the other hand, the sex pheromone of the rice looper, Plusia festucae, is composed of (Z)-5-dodecenyl acetate (Z5-12:OAc) as a main component and (Z)-7-tetradecenyl acetate (Z7-14:OAc) as a minor component (Ando et al., 1995). Both components include a double bond at the ω7-position, and the female moths produced no pheromone components unsaturated at the ω5-position. This exceptional Plusiinae pheromone is noteworthy, and we are interested in the biosynthetic pathway of the ω7-unsaturated components. Based on the information on the desaturation of many lepidopteran sex pheromones (Jurenka, 2004), we speculated that the P. festucae pheromone is produced by Δ9-desaturation of 16:Acyl or Δ11-desaturation of 18:Acyl. To clarify the pathway in P. festucae, we analyzed fatty acids in the pheromone glands by GC–MS at the start of the study. Next, deuterated C₁₆ and C₁₈ saturated and monoenoic fatty acids were treated to the pheromone glands, and the manner in which they were incorporated into Z5-12:OAc was compared. The results suggested that both desaturation made a contribution. The incorporation was also examined with a pheromone gland pretreated with an inhibitor of chain elongation to eliminate the possibility of the conversion of 16:Acid into 18:Acid. Furthermore, a similar biosynthetic experiment was carried out using another Plusiinae species, Chrysodeixis eriosoma, which secretes Z7-12:OAc as a main pheromone component (Komoda et al., 2000). Incorporation of deuterium was observed only in C. eriosoma females treated with the C₁₆ acids, indicating different β-oxidation systems in P. festucae and C. eriosoma females.

In Lepidoptera, biosynthesis of Type I pheromones has been studied with several species and the results have proposed the following pathway operating in pheromone glands; formation of saturated fatty acyl compounds from acetyl CoA → desaturation → chain shortening or elongation → reduction of the acyl moiety to a hydroxyl group → acetylation of alcohols or oxidation to aldehydes (Jurenka, 2004). Pheromone glands usually include unsaturated fatty acyl intermediates, and they have been detected by GC–MS analysis of FAMEs from lipids in the glands. For example, Z11-16:Me and Z9-14:Me were found in FAMEs derived from lipids in T. ni females in addition to Z7-12:Me, which possessed the same chain structure as the main pheromone component (Z7-12:OAc; Bjostad and Roelofs, 1983; Roelofs and Bjostad, 1984). The key step for the biosynthesis in T. ni is Δ11-desaturation (ω5-desaturation) of 16:Acyl, and Z11-16:Me is the main ester derived from the pheromone gland lipids. Z11-16:Me was not the longest one among the esters of monoenoic acids. A trace amount of the ester of a C₁₈ acid unsaturated at the ω5-position (Z13-18:Me) was detected and estimated to be produced by chain elongation of a Z11-16:Acyl intermediate.

On the other hand, a series of acyl intermediates unsaturated at the ω7-position was found in the lipids of the P. festucae females, which secreted Z5-12:OAc as a main pheromone component (Figure 2). Z9-16:Me is most abundantly included among the esters unsaturated at the ω7-position and the ratio of Z9-16:Me to Z11-16:Me detected in P. festucae is higher than those of other Plusiinae species, C. eriosoma and A. peponis (Table 1), which secrete Z7-12:OAc as a main pheromone component, as T. ni does. The data suggest that Z9-16:Acyl, which is produced by Δ9-desaturation (ω7-desaturation) of 16:Acyl, is a key biosynthetic intermediate of Z5-12:OAc. The content of Z11-18:Me is very small in P. festucae, and the ratio of Z11-18:Me to Z9-18:Me detected in P. festucae was also smaller than those in the other two Plusiinae species (Table 1). We speculated that Z11-18:Me might not be derived from a key biosynthetic intermediate of P. festucae, similarly to Z13-18:Me found in the FAMEs of T. ni females.

The biosynthetic pathway of Z5-12:OAc was confirmed by experiments with labeled fatty acids (Table 2). By topical application of D₃-16:Acid and D₁₃-Z9-16:Acid, D₃-Z5-12:OAc and D₁₃-Z5-12:OAc were respectively produced in the pheromone glands of P. festucae. The incorporation ratio of D₁₃-Z9-16:Acid was higher than that of D₃-16:Acid, supporting the desaturation step of 16:Acyl. Moreover, D₃-18:Acid and D₁₃-Z11-18:Acid were also incorporated into Z5-12:OAc. The incorporation ratios of D₃-16:Acid and D₃-18:Acid were not significantly different, and those of D₁₃-Z9-16:Acid and D₁₃-Z11-18:Acid are also similar. These results indicate that the above speculation for Z11-18:Me is incorrect. The very low content of Z11-18:Me suggests the rapid β-oxidation of Z11-18:Acyl. Thus, we concluded that not only Δ9-desaturation of 16:Acyl but also Δ11-desaturation of 18:Acyl are at work in the pheromone biosynthesis in the P. festucae females, as shown in Figure 4, if D₃-16:Acid is not easily converted into D₃-18:Acid in the pheromone glands. Since this chain elongation seems to be a common reaction, we examined the incorporation of D₃-16:Acid using females pretreated with 2-hexadecynoic acid. This acetylene compound is a known inhibitor of chain elongation of 16:Acid (Wood and Lee, 1981). The inhibitor did not affect the incorporation in P. festucae, indicating that D₃-16:Acid was desaturated without elongation and D₃-Z9-16:Acid produced from D₃-16:Acid was converted into D₃-Z5-12:OAc (Figure 4).

As a comparative study, the biosynthesis of Z7-12:OAc in C. eriosoma was examined with labeled fatty acids (Table 2). The incorporation ratios of the deuterated C₁₆ acids demonstrated the Δ11-desaturation of 16:Acyl as a key step similar to T. ni and other Plusiinae species (Komoda et al., 2000). On the contrary, deuterated C₁₈ acids were not incorporated into the pheromone. Chain shortening of the C₁₈ acids did not proceed in the pheromone gland of C. eriosoma, and it was interestingly revealed that the β-oxidation systems of P. festucae and C. eriosoma were different. The ability of β-oxidation of the C₁₈ chain is not necessary for the C. eriosoma females and the inability might fit their pheromone biosynthesis. The silkworm (Bombyx mori) secreted C₁₆ dienyl alcohol (bombykol) as a sex pheromone. Among a series of labeled fatty acids with a saturated C₁₂-C₁₈ chain, only 16:Acid was reliably incorporated into the bombykol (Ando et al., 1986). The other free acids did not enter into the biosynthetic pathway of bombykol. Although it is difficult to generalize, chain shortening and elongation of exogenous free acids do not seem to happen easily in the B. mori females. If the enzymes in the pheromone glands of P. festucae perform only a minimum necessary function, our experiment with the labeled acids clearly revealed their contribution to both types of desaturation in pheromone production.

In Plusiinae, Thysanoplusia intermixta females secrete a mixture of Z7-12:OAc and (5E,7Z)-5,7-dodecadienyl acetate (E5,Z7-12:OAc) for mating communication (Ando et al., 1998). The biosynthesis of the dienyl component was also investigated with several deuterated acids (Ono et al., 2002). While the double bond at the 5-position in the monoenyl pheromone of P. festucae is introduced at an early stage of the biosynthesis, probably just after construction of C₁₆ and C₁₈ saturated fatty acids, desaturation at the same position in the dienyl pheromone of T. intermixta is achieved on Z7-12:Acyl, which is produced by Δ11-desaturation of 16:Acyl and β-oxidation of the produced Z11-16:Acyl intermediate. This Δ5-desaturation is, in other words, ω7-desaturation. Some species in Plusiinae secrete pheromone components produced by ω3-desaturation, such as Z9-12:OAc of C. eriosoma and Z7-10:OAc of Diachrysia chrysitis (Löfstedt et al., 1994). In these species, Δ13-desaturation of 16:Acyl and Δ11-desaturation of 14:Acyl might proceed. Experimental proof for the actual substrate of ω3-desaturase is anticipated.

Whereas some Plusiinae species produce more than 10 components, only two or three compounds are essential for male attraction. Based on the chemical structures of these primary components, we have classified Plusiinae pheromones into nine groups (Ando et al., 2004; Inomata et al., 2005). All monoenyl components include the double bond at the ω3-, ω5-, or ω7-position; thus, the pheromones are produced by a combination of one of three desaturases and specific β-oxidation enzyme(s). Genes of the desaturases expressed specifically in a pheromone gland have been characterized from more than 20 lepidopteran species (Jurenka, 2004; Wang et al., 2010; Fujii et al., 2011). While the first identification of the desaturase for pheromone biosynthesis of moths was accomplished with T. ni (Knipple et al., 1998), the enzymes have unfortunately not been identified from any other Plusiinae species. In addition to providing insights on the chemical structures and biosynthetic pathways of the sex pheromones, systematic studies of the enzymes acting in the Plusiinae species would clarify speciation of this subfamily. The Plusiinae pheromones might be one of the most profitable targets for gaining an understanding of the evolution of moth species, and, therefore, further comprehensive studies are anticipated.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.