Analysis of the agrotis segetum pheromone gland transcriptome in the light of Sex pheromone biosynthesis

Abstract

BackgroundMoths rely heavily on pheromone communication for mate finding. The pheromone components of most moths are modified from the products of normal fatty acid metabolism by a set of tissue-specific enzymes. The turnip moth, Agrotis segetum uses a series of homologous fatty-alcohol acetate esters ((Z)-5-decenyl, (Z)-7-dodecenyl, and (Z)-9 tetradecenyl acetate) as its sex pheromone components. The ratio of the components differs between populations, making this species an interesting subject for studies of the enzymes involved in the biosynthetic pathway and their influence on sex pheromone variation.ResultsIllumina sequencing and comparative analysis of the transcriptomes of the pheromone gland and abdominal epidermal tissue, enabled us to identify genes coding for putative key enzymes involved in the pheromone biosynthetic pathway, such as fatty acid synthase, β-oxidation enzymes, fatty-acyl desaturases (FAD), fatty-acyl reductases (FAR), and acetyltransferases. We functionally assayed the previously identified ∆11-desaturase [GenBank: ES583599, JX679209] and FAR [GenBank: JX679210] and candidate acetyltransferases (34 genes) by heterologous expression in yeast. The functional assay confirmed that the ∆11-desaturase interacts with palmitate and produces (Z)-11-hexadecenoate, which is the common unsaturated precursor of three homologous pheromone component acetates produced by subsequent chain-shortening, reduction and acetylation. Much lower, but still visible, activity on 14C and 12C saturated acids may account for minor pheromone compounds previously observed in the pheromone gland. The FAR characterized can operate on various unsaturated fatty acids that are the immediate acyl precursors of the different A. segetum pheromone components. None of the putative acetyltransferases that we expressed heterologously did acetylate any of the fatty alcohols tested as substrates.ConclusionsThe massive sequencing technology generates enormous amounts of candidate genes potentially involved in pheromone biosynthesis but testing their function by heterologous expression or gene silencing is a bottleneck. We confirmed the function of a previously identified desaturase gene and a fatty-acyl reductase gene by heterologous expression, but the acetyltransferase postulated to be involved in pheromone biosynthesis remains illusive, in spite of 34 candidates being assayed. We also generated lists of gene candidates that may be useful for characterizing the acetyl-CoA carboxylase, fatty acid synthetase and β-oxidation enzymes.

Identification of putative Type-I sex pheromone biosynthesis-related genes expressed in the female pheromone gland of Streltzoviella insularis

“…Herein, we identified a single PBANR in the S. insularis PG transcriptome that is 84% identical to Mamestra brassicae PBANR isoform B (ARO85772.1) and is very low in abundance (0.56 FPKM; Table 2 and S1 Text). The number of PBANR-encoding genes in the S. insularis PG was in accordance with Plutella xylostella [25], Agrotis segetum [58], and Agrotis ipsilon [59]. In addition, previous studies identified three isoforms of PBANR in Ostrinia nubilalis [60] and Mamestra brassicae [61].…”

Identification and functional characterization of the pheromone biosynthesis activating neuropeptide receptor isoforms from Mamestra brassicae

“…2). All three Mambr-PBANRs clustered with high bootstrap support with PBANR sequences from other noctuid species including Spodoptera littoralis (Zheng et al, 2007), H. virescens (Kim et al, 2008), S. exigua (Cheng et al, 2010), Pseudaletia separata, H. zea, H. armigera (Lee et al, 2012) and Agrotis segetum (Ding and Löfstedt, 2015). Furthermore, all of the receptors clustered in species specific clades.…”

“…Subsequent confirmation of this hypothesis facilitated homology-based identification of the PBANR in Helicoverpa zea (Choi et al, 2003) and Bombyx mori (Hull et al, 2004). Since then PBANRs have been identified in diverse moth (Cheng et al, 2010;Ding and Löfstedt, 2015;Kim et al, 2008;Lee et al, 2012;Nusawardani et al, 2013;Rafaeli et al, 2007;Zheng et al, 2007) with multiple PBANR isoforms (PBANR-As, -A, -B, and -C) shown to be concomitantly expressed in PGs (Kim et al, 2008;Lee et al, 2012). The isoforms are differentiated by the length and composition of their C-termini, which is necessary for the ligand-induced internalization (Hull et al, 2005), a phase of GPCR feedback regulation and desensitization (Moore et al, 2007;Marchese et al, 2008).…”

Biotechnological potential of insect fatty acid-modifying enzymes

“…Although moth FARs usually exhibit a broad substrate preference [77,89,111,120,122,123], some of them display specificity to unsaturated substrates with a double bond in either the E or Z configuration [119], a particular chain length and double bond position [120], or a system of conjugated double bonds [118]. Given the multiple FAR paralogs generally present in insect genomes [13,14,16,17,111,124] and limited knowledge of their properties, there is a demand to perform functional characterization of these biologically and biotechnologically relevant enzymes. FARs from insects other than moths are virtually unexplored.…”

“…The diversification of the pheromone-biosynthetic enzymes likely has been driven by evolutionarily imposed requirements on sex pheromone signal specificity [11,12]. A broad spectrum of insect pheromone-biosynthetic enzymes has already been functionally characterized, and transcriptomic sequencing (RNA-seq) of pheromone glands using next-generation sequencing has identified new candidates for characterization [13][14][15][16][17][18][19]. RNA-seq of other insect glands and tissues may open the door to the discovery of many additional enzymes with remarkable biosynthetic capabilities [8].…”