454 pyrosequencing based transcriptome analysis of Zygaena filipendulae with focus on genes involved in biosynthesis of cyanogenic glucosides

Abstract

Background: An essential driving component in the co-evolution of plants and insects is the ability to produce and handle bioactive compounds. Plants produce bioactive natural products for defense, but some insects detoxify and/or sequester the compounds, opening up for new niches with fewer competitors. To study the molecular mechanism behind the co-adaption in plant-insect interactions, we have investigated the interactions between Lotus corniculatus and Zygaena filipendulae. They both contain cyanogenic glucosides which liberate toxic hydrogen cyanide upon breakdown. Moths belonging to the Zygaena family are the only insects known, able to carry out both de novo biosynthesis and sequestration of the same cyanogenic glucosides as those from their feed plants. The biosynthetic pathway for cyanogenic glucoside biosynthesis in Z. filipendulae proceeds using the same intermediates as in the well known pathway from plants, but none of the enzymes responsible have been identified. A genomics strategy founded on 454 pyrosequencing of the Z. filipendulae transcriptome was undertaken to identify some of these enzymes in Z. filipendulae.

Results: Comparisons of the Z. filipendulae transcriptome with the sequenced genomes of Bombyx mori, Drosophila melanogaster, Tribolium castaneum, Apis mellifera and Anopheles gambiae indicate a high coverage of the Z. filipendulae transcriptome. 11% of the Z. filipendulae transcriptome sequences were assigned to Gene Ontology categories. Candidate genes for enzymes functioning in the biosynthesis of cyanogenic glucosides (cytochrome P450 and family 1 glycosyltransferases) were identified based on sequence length, number of copies and presence/absence of close homologs in D. melanogaster, B. mori and the cyanogenic butterfly Heliconius. Examination of biased codon usage, GC content and selection on gene candidates support the notion of cyanogenesis as an "old" trait within Ditrysia, as well as its origins being convergent between plants and insects.

Conclusion: Pyrosequencing is an attractive approach to gain access to genes in the biosynthesis of bio-active natural products from insects and other organisms, for which the genome sequence is not known. Based on analysis of the Z. filipendulae transcriptome, promising gene candidates for biosynthesis of cyanogenic glucosides was identified, and the suitability of Z. filipendulae as a model system for cyanogenesis in insects is evident.

Figure 1

Proposed biosynthetic pathway for the CNglcs linamarin and lotaustralin. The types of enzymes catalyzing the equivalent reactions in plants are shown. Intermediates known from insects are indicated with a star.

Figure 2

Distribution of sequence lengths and cluster sizes. A: Number distribution of lengths of the individual reads. B: Number distribution of lengths of the contigs. Average contig length is indicated by a green line. C: Contig sizes. Contig sizes are scaling invariant as observed by [17].

Figure 3

Gene ontology (GO) -terms for Z. filipendulae proteins belonging to match level 5. The percentage, (total number), and distribution of top-level GO-terms for match level 5 and better in the categories "Molecular function", "Biological Process" and "Cellular component". Total number of Z. filipendulae genes in each category is also shown.

Figure 4

A comparison of the distribution of sequence identity of Z. filipendulae proteins to proteins from five different insect species. Numerical distribution of protein sequence identity between Z. filipendulae protein sequences obtained, and the best BLASTp search hit when compared to available B. mori, D. melanogaster, T. castaneum, A. mellifera, and A. gambiae protein sequences. All curves are normalized so that the area beneath each curve sums to 1. The drop in matches with less than roughly 25% identity is probably an artifact of the BLAST E-value threshold.

Figure 5

Phylogenetic relationships and genetic overlap between Z. filipendulae, B. mori and D. melanogaster. A: Simplified evolutionary tree showing the relationships between Zygaena, Heliconius, Bombyx, Drosophila, Anopheles, Apis, and Tribolium. Groups comprising cyanogenic species are shown in bold. Time since divergence of the lineages Papilionoidea/Bombycoidea and Lepidoptera/Diptera are shown in italics [20]. B: Venn diagram of the number of contigs (upper number) and singlets (lower number) from Z. filipendulae which show matches to D. melanogaster (19,389 proteins) and/or B. mori (14,623 proteins). Cut-off E-value was 1e^-3.

Figure 6

Neighbor-Joining bootstrap tree of translated full length Z. filipendulae P450 sequences. Sequences from Z. filipendulae are marked in red. Clades of insect P450s are according to [26,27]. Bm: B. mori, Dm: D. melanogaster, EP: E. postvittana. Bootstrap values are shown as percentages.

Figure 7

Neighbor-Joining bootstrap tree of translated full length Z. filipendulae UGT sequences . Sequences from Z. filipendulae are marked in red. Aa:Aedes aegypti, Bm:B. mori, Cq:Culex quinquefasciatus, Sf:S. frugiperda, Tc:T. castaneum. Bootstrap values are shown as percentages.