Expansion of cytochrome P450 and cathepsin genes in the generalist herbivore brown marmorated stink bug

Abstract

Background: The brown marmorated stink bug (Halyomorpha halys) is an invasive pest in North America which causes severe economic losses on tree fruits, ornamentals, vegetables, and field crops. The H. halys is an extreme generalist and this feeding behaviour may have been a major contributor behind its establishment and successful adaptation in invasive habitats of North America. To develop an understanding into the mechanism of H. halys' generalist herbivory, here we specifically focused on genes putatively facilitating its adaptation on diverse host plants.

Results: We generated over 142 million reads via sequencing eight RNA-Seq libraries, each representing an individual H. halys adult. The de novo assembly contained 79,855 high quality transcripts, totalling 39,600,178 bases. Following a comprehensive transcriptome analysis, H. halys had an expanded suite of cytochrome P450 and cathepsin-L genes compared to other insects. Detailed characterization of P450 genes from the CYP6 family, known for herbivore adaptation on host plants, strongly hinted towards H. halys-specific expansions involving gene duplications. In subsequent RT-PCR experiments, both P450 and cathepsin genes exhibited tissue-specific or distinct expression patterns which supported their principal roles of detoxification and/or digestion in a particular tissue.

Conclusions: Our analysis into P450 and cathepsin genes in H. halys offers new insights into potential mechanisms for understanding generalist herbivory and adaptation success in invasive habitats. Additionally, the large-scale transcriptomic resource developed here provides highly useful data for gene discovery; functional, population and comparative genomics as well as efforts to assemble and annotate the H. halys genome.

Keywords: Adaptation; Generalist; Halyomorpha halys; Herbivory; Invasion.

Fig. 1

Halyomorpha halys de novo assembly. a Length distribution of 79,855 transcript contigs in de novo assembly (b) Ortholog hit ratio for transcripts calculated after blastx searches to genomes of A. pisum, B. mori, D. melanogaster, N. vitripennis, R. prolixus, and T. castaneum (c) Distribution of E values for top hits obtained during blastx search is shown

Fig. 2

Summary of top hit organisms in blastx search for Halyomorpha halys transcripts. Overall distribution for the whole transcriptome is shown on the left whereas insect specific distributions are presented on right

Fig. 3

Comparative genomics for Halyomorpha halys transcriptome. a Venn diagram showing the number of transcript contigs with significant matches (unique and common) to genomes of A. pisum, D. melanogaster, R. prolixus, and T. castaneum. Significant matches (e value <1.0E-3) were calculated after pairwise comparisons (blastx) to each individual genome. b Comparison of GO term mappings distributions of H. halys and A. pisum that belong to each of the three top-level GO categories (i.e. biological process, molecular function, and cellular component)

Fig. 4

Classification of cytochrome P450 genes in Halyomorpha halys. Clan and family distribution of P450 genes in H. halys is shown. The number shown along each column represents the P450 family and the number in parenthesis is the number of individual genes predicted in the corresponding family

Fig. 5

Phylogenetic analysis of P450s in Halyomorpha halys. A phylogenetic tree generated using P450s of CYP6 family in H. halys and other insects is shown. The evolutionary history was inferred by using the Maximum Likelihood method based on the Le Gascuel model. All nodes have significant bootstrap support based on 500 replicates. The bootstrap values only above 50% are shown next to branches. GenBank accession numbers for various protein sequences used in the phylogenetic analysis are provided in Additional file 2

Fig. 6

Tissue expression analysis for P450 genes in Halyomorpha halys. Results of semi quantitative PCR for expression analysis of CYP6BQ genes in H. halys gut, salivary gland (SG), fat body (FB), malpighian tubule (MT), and ovary (OV) tissues are shown (gel panels A-D; from left). Numbers on left for each gel section indicate P450 contig ID in the transcriptome assembly. HhEF1a was used as internal control. Primers and contig sequences are provided in Additional file 1

Fig. 7

Amino acid alignment of cathepsins in Halyomorpha halys. Amino acid alignment of a few selected cathepsin-L proteases is shown. Functionally important residues C, H, and N (active sites) are boxed (indicated by arrows)

Fig. 8

Phylogenetic analysis of cathepsins in Halyomorpha halys. A phylogenetic tree generated using cathepsins (Cathepsin-B and Cathepsin-L) in H. halys along with corresponding cathepsins in other insects is shown. The evolutionary history was inferred by using the Maximum Likelihood method based on the Whelan and Goldman model. All nodes have significant bootstrap support based on 1000 replicates. The bootstrap values only above 50% are shown next to branches. The asterisks indicate the locations of H. halys cathepsin-L gene expansion. GenBank accession numbers for various protein sequences used in the phylogenetic analysis are provided in Additional file 2

Fig. 9

Tissue expression analysis for cathepsin-L genes in Halyomorpha halys. Results of semi quantitative PCR for expression analysis of cathepsin-L genes in H. halys gut, salivary gland (SG), fat body (FB), malpighian tubule (MT), and ovary (OV) tissues are shown (gel panels A-E; from left). HhEF1a was used as internal control. Primers sequences are provided in Additional file 1