Transcriptome sequencing of Tessaratoma papillosa antennae to identify and analyze expression patterns of putative olfaction genes

Abstract

Studies on insect olfaction have increased our understanding of insect's chemosensory system and chemical ecology, and have improved pest control strategies based on insect behavior. In this study, we assembled the antennal transcriptomes of the lychee giant stink bug, Tessaratoma papillosa, by using next generation sequencing to identify the major olfaction gene families in this species. In total, 59 odorant receptors, 14 ionotropic receptors (8 antennal IRs), and 33 odorant binding proteins (28 classic OBPs and 5 plus-C OBPs) were identified from the male and female antennal transcriptomes. Analyses of tissue expression profiles revealed that all 59 OR transcripts, 2 of the 8 antennal IRs, and 6 of the 33 OBPs were primarily expressed in the antennae, suggesting their putative role in olfaction. The sex-biased expression patterns of these antenna-predominant genes suggested that they may have important functions in the reproductive behavior of these insects. This is the first report that provides a comprehensive resource to future studies on olfaction in the lychee giant stink bug.

Figure 1

(A) Unigenes annotated via different databases. (B) Percentage of top hits from other insect species homologous to T. papillosa transcripts. Transcripts from T. papillosa were searched by BLASTx against the non-redundant protein database with a cutoff E-value of 10⁻⁵. (C) Gene ontology (GO) classification of T. papillosa transcripts using Blast2GO. One unigene was annotated to more than one GO term.

Figure 2

Phylogenetic analysis of putative T. papillosa ORs with other hemipteran ORs. The dendrogram was generated by FastTree2 (JTT substitution model). Species abbreviations: Tpap, Tessaratoma papillosa; Aluc, Apolygus lucorum; Hhal, Halyomorpha halys; Sfur, Sogatella furcifera. Amino acid sequences used for tree construction are shown in Supplementary Table S2. Branch support (circles at the branch nodes) was estimated using an approximate likelihood ratio test based on the scale indicated at the top left. Bars indicate branch lengths in proportion to amino acid substitutions per site.

Figure 3

Relative expression levels of putative T. papillosa ORs in the female and male antennae, and other body parts. Abbreviations: FA, female antennae; MA, female antennae; Bo, other body parts (the pooled tissue mixture of proboscis, stink gland, midgut, foreleg tarsus, and wing). The expression levels were estimated using the 2^−ΔΔCT method. The relative expression level is indicated as mean ± SE (n = 4). Standard error is represented by the error bar, and different letters indicate statistically significant difference between tissues (p < 0.05, ANOVA, HSD).

Figure 4

(A) Phylogenetic analysis of putative T. papillosa iGluRs/IRs in with other insect iGluRs/IRs. The dendrogram was generated by FastTree2 (JTT substitution model). Species abbreviations: Tpap, Tessaratoma papillosa; Apis, Acyrthosiphon pisum; Bmor, Bombyx mori; Dmel, Drosophila melanogaster. Amino acid sequences used to construct the tree are shown in Supplementary Table S2. Branch support (circles at the branch nodes) was estimated using an approximate likelihood ratio test based on the scale indicated at the top left. Bars indicate branch lengths in proportion to amino acid substitutions per site. (B) Excerpts from the amino acids alignment showing the predicted iGluRs/IRs binding domains.

Figure 5

(A) Transcriptional profiles of putative T. papillosa iGluRs/IRs in different body parts as determined by semi-quantitative RT-PCR. Red dots represent iGluRs/IRs that are upregulated in the antenna. Two reference genes labeled with blue triangle, ubiquitin conjugation factor E4 A (TpapUBE4A) and 60 S ribosomal protein L32 (TpapRPL32), were used as internal references to test the integrity of each cDNA template. Abbreviations: A: antenna; P, proboscis; SG, stink gland; MG, midgut; T, tarsus. (B) Relative expression levels of antenna-predominant iGluRs/IRs in the female and male antennae, and other body parts. Abbreviations: FA, female antennae; MA, female antennae; Bo, other body parts (the pooled tissue mixture of proboscis, stink gland, midgut, foreleg tarsus, and wing). The expression levels were estimated using the 2^−ΔΔCT method. Relative expression level is indicated as mean ± SE (n = 4). Standard error is represented by the error bar, and different letters indicate statistically significant difference between tissues (p < 0.05, ANOVA, HSD).

Figure 6

(A) Phylogenetic analysis of putative T. papillosa OBPs with other hemipteran OBPs. Plus-C OBPs from T. papillosa and other putative hemipterans form a clade labeled in dark green. Labeled in orange are classic OBPs from T. papillosa and other putative hemipterans. Species abbreviations: Tpap, Tessaratoma papillosa; Hhal, Halyomorpha halys; Llin, Lygus lineolaris; Aluc, Apolygus lucorum. Alin, Adelphocoris lineolatus, Asut, Adelphocoris suturalis, Save, Sitobion avenae, Apis, Acyrthosiphon pisum, Agos, Aphis gossypii, Nlug, Nilaparvata lugens, Sfur, Sogatella furcifera, Dcit, Diaphorina citri. Amino acid sequences used to construct the tree are shown in Supplementary Table S2. (B) Transcriptional profiles of putative T. papillosa OBPs in different body parts determined by semi-quantitative RT-PCR. The OBPs that are upregulated in the antennae are labeled with red dots. Two reference genes labeled with blue triangle, ubiquitin conjugation factor E4 A (TpapUBE4A) and 60 S ribosomal protein L32 (TpapRPL32), were used as internal references to test the integrity of each cDNA templates. Abbreviations: A: antenna; P, proboscis; SG, stink gland; MG, midgut; T, tarsus. (C) Relative expression levels of antenna-predominant OBPs in the female and male antennae, and other body parts. Abbreviations: FA, female antennae; MA, female antennae; Bo, other body parts. Expression levels were estimated using the 2^−ΔΔCT method. The relative expression level is indicated as mean ± SE (n = 4). Standard error is represented by the error bar, and different letters indicate statistically significant difference between tissues (p < 0.05, ANOVA, HSD).