Transcriptome analysis of the sex pheromone gland of the noctuid moth Heliothis virescens

Abstract

Background: The 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.

Results: To 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.

Conclusion: These 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.

Figure 1

Dissection of Hv sex pheromone gland for RNA extraction. A. Gland was forced out by squeezing the abdomen with pincet (the gland is similary inflated when the female calls (Groot, Schal, Gould, Classen, pers. obs). B. Gland was cut at the scleritozed cuticle from the 8^th abdominal segment. C. Sclerotized cuticle was mostly removed before immersing gland in Trizol.

Figure 2

Venn diagram showing the total number of contigs (in black) overlapping between the three pheromone gland EST databases. Percentages are given of the relative number of overlapping contigs compared to the total number of contigs found in the pheromone gland (colors match the species colors).

Figure 3

All pie charts combined into a bar graph; overview of GO-level 3. Note that one gene object can be classified into more than 1 class, therefore the total number of gene objects classified for Hv-PGN is not 2451 (2501 - 50), but 5060, indicating that on average one contig is classified into 2 classes. Asterisks denote absence in one of the libraries in the respective GO category.

Figure 4

Overlapping gene objects (in grey) possibly involved in the biosynthetic pathway of sex phermone production when comparing the different libraries (categories the same as in Figure 5). HvPG_minus_HvLN: ESTs in HvPG that were not found in HvLN. HvPG_minus_HvLN_BmPG: ESTs in HvPG that were not found in HvLN, but were found in the BmPG library. HvPG_BmPG: ESTs that were found both in HvPG and in BmPG. HvPG_AsPG: ESTs that were found both in HvPG and in AsPG. HvPG_BmPG_AsPG: ESTs that were found in HvPG, BmPG and AsPG.

Figure 5

Proposed biosynthetic pathway of sex pheromone production in female moths (adapted from [14,17,25].

Figure 6

Gene phylogeny and sequence similarity of Accase protein sequences. A. Neighbor-joining (NJ) consensus tree of ACCase sequences from Heliothis virescens (Hvir), Tribolium castaneum (Tcas; XP_969851), Pediculus humanus corporis (Phuc; XP_002429216), Drosophila melanogaster (Dmel; NP_610342), Danio rerio (Drer; XP_001919815) and Homo sapiens (Hsap; AJ575431). Bootstrap values from NJ analyses are shown as percentages. B. MAFFT alignment with part of Accase proteins listed in the phylogeny. Identical amino acids are shaded in black and depicted by an asterisc, conserved amino acids are shaded in grey and depicted by a dot in the consensus sequence.

Figure 7

Gene phylogeny of FAR protein sequences. Neighbor-joining (NJ) consensus tree of FAR sequences from Heliothis virescens (Hvir), Bombyx mori (Bmor; NP_001036967), Ostrinia scapulalis (Osca; ACJ06514), Tribolium castaneum (Tcas; XP_967757), Pediculus humanus corporis (Phuc; XP_002428142), and Drosophila melanogaster (Dmel; AAF46099). Bootstrap values from NJ analyses are shown as percentages of a total of 1000 bootstrap runs.

Figure 8

Overlapping gene objects (in grey) involved in pheromone perception and/or degradation when comparing the different libraries (see Figure 4 for explanation of the headings).

Figure 9

PCR products using the primers described by Widmayer et al. (2009) on the normalized and non-normalized cDNA pool of the Hv pheromone gland. Abbreviations used as in Widmayer et al: PBP: Pheromone Binding Protein; HR: Heliothis Chemosensory Receptor; RL: Ribosomal Protein.