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Analysis of the Agrotis segetum pheromone gland transcriptome in the light of sex pheromone biosynthesis.

ABSTRACT: Moths 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.Illumina 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, ?-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.The 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 ?-oxidation enzymes.

SUBMITTER: Ding BJ

PROVIDER: S-EPMC4575462 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Analysis of the Agrotis segetum pheromone gland transcriptome in the light of sex pheromone biosynthesis.

Ding Bao-Jian BJ Löfstedt Christer C

BMC genomics 20150918

<h4>Background</h4>Moths 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 interestin ...[more]

PMID: 26385554

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Project description:BackgroundOne of the challenges in insect chemical ecology is to understand how insect pheromones are synthesised, detected and degraded. Genome wide survey by comparative sequencing and gene specific expression profiling provide rich resources for this challenge. A. ipsilon is a destructive pest of many crops and further characterization of the genes involved in pheromone biosynthesis and transport could offer potential targets for disruption of their chemical communication and for crop protection.ResultsHere we report 454 next-generation sequencing of the A. ipsilon pheromone gland transcriptome, identification and expression profiling of genes putatively involved in pheromone production, transport and degradation. A total of 23473 unigenes were obtained from the transcriptome analysis, 86% of which were A. ipsilon specific. 42 transcripts encoded enzymes putatively involved in pheromone biosynthesis, of which 15 were specifically, or mainly, expressed in the pheromone glands at 5 to 120-fold higher levels than in the body. Two transcripts encoding for a fatty acid synthase and a desaturase were highly abundant in the transcriptome and expressed more than 40-fold higher in the glands than in the body. The transcripts encoding for 2 acetyl-CoA carboxylases, 1 fatty acid synthase, 2 desaturases, 3 acyl-CoA reductases, 2 alcohol oxidases, 2 aldehyde reductases and 3 acetyltransferases were expressed at a significantly higher level in the pheromone glands than in the body. 17 esterase transcripts were not gland-specific and 7 of these were expressed highly in the antennae. Seven transcripts encoding odorant binding proteins (OBPs) and 8 encoding chemosensory proteins (CSPs) were identified. Two CSP transcripts (AipsCSP2, AipsCSP8) were highly abundant in the pheromone gland transcriptome and this was confirmed by qRT-PCR. One OBP (AipsOBP6) were pheromone gland-enriched and three OBPs (AipsOBP1, AipsOBP2 and AipsOBP4) were antennal-enriched. Based on these studies we proposed possible A. ipsilon biosynthesis pathways for major and minor sex pheromone components.ConclusionsOur study identified genes potentially involved in sex pheromone biosynthesis and transport in A. ipsilon. The identified genes are likely to play essential roles in sex pheromone production, transport and degradation and could serve as targets to interfere with pheromone release. The identification of highly expressed CSPs and OBPs in the pheromone gland suggests that they may play a role in the binding, transport and release of sex pheromones during sex pheromone production in A. ipsilon and other Lepidoptera insects.

Project description:BackgroundThe chemical components of sex pheromones have been determined for more than a thousand moth species, but so far only a handful of genes encoding enzymes responsible for the biosynthesis of these compounds have been identified. For understanding the evolution of moth sexual communication, it is essential to know which genes are involved in the production of specific pheromone components and what controls the variation in their relative frequencies in the pheromone blend. We used a transcriptomic approach to characterize the pheromone gland of the Noctuid moth Heliothis virescens, an important agricultural pest, in order to obtain substantial general sequence information and to identify a range of candidate genes involved in the pheromone biosynthetic pathway.ResultsTo facilitate identifying sets of genes involved in a broad range of processes and to capture rare transcripts, we developed our majority of ESTs from a normalized cDNA library of Heliothis virescens pheromone glands (PG). Combining these with a non-normalized library yielded a total of 17,233 ESTs, which assembled into 2,082 contigs and 6,228 singletons. Using BLAST searches of the NR and Swissprot databases we were able to identify a large number of putative unique gene elements (unigenes), which we compared to those derived from previous transcriptomic surveys of the larval stage of Heliothis virescens. The distribution of unigenes among GO Biological Process functional groups shows an overall similarity between PG and larval transcriptomes, but with distinct enrichment of specific pathways in the PG. In addition, we identified a large number of candidate genes in the pheromone biosynthetic pathways.ConclusionThese data constitute one of the first large-scale EST-projects for Noctuidae, a much-needed resource for exploring these pest species. Our analysis shows a surprisingly complex transcriptome and we identified a large number of potential pheromone biosynthetic pathway and immune-related genes that can be applied to population and systematic studies of Heliothis virescens and other Noctuidae.

Project description:Lepidopteran pheromone receptors (PRs), for which orthologies are evident among closely related species, provide an intriguing example of gene family evolution in terms of how new functions may arise. However, only a limited number of PRs have been functionally characterized so far and thus evolutionary scenarios suffer from elements of speculation. In this study we investigated the turnip moth Agrotis segetum, in which female moths produce a mixture of chemically related pheromone components that elicit specific responses from receptor cells on male antennae. We cloned nine A. segetum PR genes and the Orco gene by degenerate primer based RT-PCR. The nine PR genes, named as AsegOR1 and AsegOR3-10, fall into four distinct orthologous clusters of known lepidopteran PRs, of which one contains six paralogues. The paralogues are under relaxed selective pressure, contrasting with the purifying selection on other clusters. We identified the receptors AsegOR9, AsegOR4 and AsegOR5, specific for the respective homologous pheromone components (Z)-5-decenyl, (Z)-7-dodecenyl and (Z)-9-tetradecenyl acetates, by two-electrode voltage clamp recording from Xenopus laevis oocytes co-expressing Orco and each PR candidate. These receptors occur in three different orthologous clusters. We also found that the six paralogues with high sequence similarity vary dramatically in ligand selectivity and sensitivity. Different from AsegOR9, AsegOR6 showed a relatively large response to the behavioural antagonist (Z)-5-decenol, and a small response to (Z)-5-decenyl acetate. AsegOR1 was broadly tuned, but most responsive to (Z)-5-decenyl acetate, (Z)-7-dodecenyl acetate and the behavioural antagonist (Z)-8-dodecenyl acetate. AsegOR8 and AsegOR7, which differ from AsegOR6 and AsegOR1 by 7 and 10 aa respectively, showed much lower sensitivities. AsegOR10 showed only small responses to all the tested compounds. These results suggest that new receptors arise through gene duplication, and relaxed evolutionary constraints or positive selection among paralogues allow functional divergence to occur in spite of purifying selection being the norm.

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Analysis of the Agrotis segetum pheromone gland transcriptome in the light of sex pheromone biosynthesis.

Publications

Analysis of the Agrotis segetum pheromone gland transcriptome in the light of sex pheromone biosynthesis.

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