Overview of the PG transcriptome
The PG transcriptome of E. hippophaecolus was sequenced and analyzed. Among the identified 20,363 unigenes, only 50.60% were annotated to one or more GO terms, which indicated that massive E. hippophaecolus genes are either non-coding or share homology with genes not associated with GO terms, similar to the results of transcriptome analysis of the sex PG of Spodoptera litura [47], H. armigera and H. assulta [7], Plutella xylostella [48], Ephestia cautella [49], Agrotis ipsilon [50], Lutzomyia longipalpis [51], Heliothis virescens [52], and Agrotis segetum [53]. What's more, we identified 77 putative genes refered to sex pheromone biosynthesis in E. hippophaecolus, significantly more than the 55 identified in S. litura, [47], but fewer than the 87 in P. xylostella [48]. We also identified 31 chemoreception proteins in E. hippophaecolus, of which 13 were novel [46], and 11 ODEs. All in all, the results not merely offer a vital foundation for further elucidation of the molecular mechanisms of sex pheromone synthesis and metabolism, but also afford general perception into insect physiology and the exploitation of novel pest control strategies [54].
Genes involved in the sex pheromone biosynthesis pathway
Pheromone biosynthesis-activating neuropeptide receptor (PBANr)
In the PG of E. hippophaecolus, we identified two PBANr and two PBAN genes. Only four alternative splice transcripts of PBANr isoforms (A, B, and C) with diversities in C-terminal sequences have been reported for a single insect species [55]. PBAN receptor isoforms B and C have been characterized from O. nubilalis [56] and H. virescens [57] functionally, and isoforms C and B were identified in the PG of E. hippophaecolus in the present work, both presumably involved in pheromone production. Analysis of the PG transcriptomes of A. segatum [58] and H. virescens [52] revealed a G protein gamma subunit homolog, the diapause hormone receptor, that interacts with PBANr.
Acetyl-CoA carboxylase (ACC)
ACC catalyzing the production of malonyl-CoA from acetyl-CoA is the first committed and rate restrictive biosynthetic step in start pheromone biosynthesis [10, 59]. In the E. hippophaecolus PG, we identified five transcripts encoding ACCs expressed at relatively low levels.
Fatty acid synthase (FAS)
FAS is believed to facilitate the transformation of malonyl-CoA and NADPH to generate saturated fatty acids (16:acyl in E. hippophaecolus) [49]. We identified six FAS unigenes in the E. hippophaecolus PG, among which EhipFAS3 has the longest open reading frame (ORF) and is expressed at distinct high levels (FPKM = 913), suggesting that it may be the main FAS.
Fatty acid transport protein (FATP)
The evolutionarily conserved membrane-bound proteins family FATPs promote the ingestion of extracellular long-chain fatty acids (LCFAs), and/or very LCFAs, and facilitate the ATP-dependent esterification of these fatty acids to their corresponding acyl-CoA derivatives [60]. In E. hippophaecolus, we identified one FATP, the function of which has been showed by in vitro expression and knockdown analysis in B. mori [60] and Eilema japonica [61].
Acyl-CoA desaturase (DES)
Moth sex pheromones typically include double bonds introduced by desaturases at specific locations in the fatty acyl carbon chains [25, 62, 63]. Newly in some Lepidoptera species, several reductase gene family have been discovered and functionally characterized, including Heliothis virescens, Heliothis subflexa, H. armigera and H. assulta [65], Ostrinia scapulalis [64], Yponomeuta evonymellus (L.), Yponomeuta padellus (L.), and Yponomeuta rorellus (Hübner) [31], and Ostrinia nubilalis [66]. Sixteen putative sex pheromone components extracted from the E. hippophaecolus PG were unsaturated fatty acids with £16 carbons and acetate as the functional group. Notably, Z7-14:Ac and E3-14:Ac were previously identified as pheromones in E. hippophaecolus [44], and C16 fatty acids require β-oxidation to form short-chain C14 fatty acids, but β-oxidation enzymes were not identified in the E. hippophaecolus transcriptome. Female sex pheromones in the E. hippophaecolus PG have been identified as Z7-14:Ac and E3-14:Ac [44]. It is therefore rational to suggest that the saturated fatty acid precursor of E. hippophaecolus sex pheromones may be palmitic acid (16:0), which is desaturated by Δ11-desaturase to constitute the precursor Z11-16:acyl-CoA for the production of two major components (Z7-14:OAc and E3-14:OAc; Figure 7). Other studies in Lepidoptera species indicate that a Δ11-desaturase acting on palmitic acid leads to the production of the sex pheromone components [67, 68]. In the E. hippophaecolus PG transcriptome, we identified three Δ11-desaturases, three Δ9-desaturases, and one Δ4-desaturase, and based on the DES phylogenetic tree, EhipDES3 is another Δ11-desaturase named EhipΔ11DES-4.
Fatty acyl-CoA reductase (FAR)
Through a specific Δ11 double bond brought in fatty acid precursors a fatty acyl-CoA precursor was formed, which is then shortened sequentially by β-oxidation for formation different shorter chain fatty acyl-CoA precursors [1] that are further lessened separately by FAR for formation corresponding fatty alcohols [31, 69]. We identified 19 FAR unigenes. Since the discovery of their ability to reduce fatty acids to alcohols in B. mori [19], these enzymes have also been functionally characterized in Ostrinia spp. [28, 66], Yponomeuta spp. [31], Helicoverpa spp., and Heliothis spp. [65]. FARs from Spodoptera exigua exhibit a preference for C14 and C16 fatty acid substrates [70].
Acetyltransferase (ACT)
In E. hippophaecolus, sex pheromone mixtures only contain acetates which are intermediates that are acetylated to acetate esters by ACTs [71]. In the present study, we identified 12 ACT unigenes. However, ACT enzymes that have not been verified functionally are postulated to be participated in the sex pheromone biosynthesis pathway. We identified only one ACBP gene, and its counterpart is expressed in the B. mori PG mainly, suggesting that ACBP takes vital effect on the production of sex pheromones adjusted by the neurohormone PBAN [72]. The proposed biosynthesis pathway for Z7-14:Ac and E3-14:Ac in E. hippophaecolus is shown in Figure 7.
Genes involved in chemoreception
Moth ovipositors have chemoreceptive sensilla [73, 74], and the sex PG expresses chemoreception proteins such as OBPs and CSPs that are postulated to facilitate the transportation of sex pheromones. OBPs are in the first gate of the odorant recognition process, which combine and transport especially for hydrophobic odorant molecules, including plant volatiles and pheromones across the lymph in the sensillum [75]. We identified 16 OBPs, including three new genes not previously identified in the antennae of E. hippophaecolus, in which EhipOBP2, EhipOBP4, EhipOBP5, and EhipOBP8 are expressed in male antennae, while EhipOBP1 and EhipOBP6 are expressed in the foot and external genitals, respectively [46]. EhipPBP3 was the second most abundant of the three PBPs in male antennae at both gene and protein expression levels [76]. The subfamily of OBPs, CSPs reveal diverse expression profiles with OBPs. OBP expression is antennae-biased, but CSPs are without obvious expression preference [77]. We identified 14 OBPs, including one new CSP not previously identified, in the antennae of E. hippophaecolus [46]. ORs couple binding proteins with olfactory sensory neurons and conduct olfactory signal transduction. In female and male antennae, we detected three known ORs, including the pheromone receptor EhipPR2 [46]. We identified seven IRs, including four novel genes [46]. Genome-wide analysis of IRs in Heliconius butterflies has been carried out [78], and the results of dendrogram analysis were consistent with their corresponding functions. Specifically, we identified EhipIR13 and EhipIR14 in the IR75 clade, which suggests that EhipIR13 and EhipIR14 are other types of EhipIR75p1, named EhipIR75p1-1(13) and EhipIR IR75p1-2(14).Notably IR8a and IR25a are considered function as IR co-receptors [79, 80], and also identified in the PG transcriptome. We also detected six GRs, including five new genes, and two known SNMPs [46] that are conserved throughout holometabolous insects [81, 82]. IRs are a conserved family of synaptic ligand-gated ion channels that evolved from ionotropic glutamate receptors (iGluRs) [79, 80], which are refered to both insects’ smell and taste process [83, 84]. Mutations of IR64a, IR84a, IR25a, and IR8a in Drosophila restrain odorant-evoked neuronal responses [85, 86]. However, the specificity of ligand recognition by IRs is unclear [83]. Intriguingly, Drosophila IR94b is functionally involved in auditory system [87]. Five ODEs and six AES enzymes were identified in the PG transcriptome, all of which are new in E. hippophaecolus [46].
In summary, the female PG expresses OBPs, CSPs, ORs, IRs, GRs, SNMPs, and ODEs that together may mediate pheromone biosynthesis and recognition. Analysis of pheromone biosynthesis and chemoreception genes in the PG and antennae of E. hippophaecolus can reveal details of pheromone biosynthesis, chemoreception, and transport and degradation mechanisms, which may elucidate connections between pheromone biosynthesis in females and pheromone recognition in male antennae.