Transcriptome-Wide Identification of Cytochrome P450s in Tea Black Tussock Moth (Dasychira baibarana) and Candidate Genes Involved in Type-II Sex Pheromone Biosynthesis

Abstract

The tea black tussock moth (Dasychira baibarana), a devastating pest in Chinese tea plantations, uses a ternary Type-II pheromone blend containing (3Z,6Z)-cis-9,10-epoxyhenicosa-3,6-diene (Z3,Z6,epo9-21:H), (3Z,6Z,11E)-cis-9,10-epoxyhenicosa-3,6,11-triene (Z3,Z6,epo9,E11-21:H), and (3Z,6Z)-henicosa-3,6-dien-11-one (Z3,Z6-21:11-one) for mate communication. To elucidate the P450 candidates associated with the biosynthesis of these sex pheromone components, we sequenced the female D. baibarana pheromone gland and the abdomen excluding the pheromone gland. A total of 75 DbP450s were identified. Function annotation suggested six CYPs were orthologous genes that are linked to molting hormone metabolism, and eight antennae specifically and significantly up-regulated CYPs may play roles in odorant processing. Based on a combination of comparative RNAseq, phylogenetic, and tissue expression pattern analysis, one CYP4G with abdomen specifically predominant expression pattern was likely to be the P450 decarbonylase, while the pheromone-gland specifically and most abundant CYP341B65 was the most promising epoxidase candidate for the D. baibarana sex pheromone biosynthesis. Collectively, our research laid a valuable basis not only for further functional elucidation of the candidate P450 decarbonylase and epoxidase for the sex pheromone biosynthesis but also for understanding the physiological functions and functional diversity of the CYP gene superfamily in the D. baibarana.

Keywords: RNAseq; Type-II sex pheromone; biosynthesis; cytochrome P450s; phylogenetic analysis; tea black tussock moth.

Figure 1

The gene ontology (GO) classification. All unigenes can be classified into one or more categories. The number in each sector represents the total number of unigenes in each category. The analysis was at level 3.

Figure 2

Expression levels of the identified P450s in the tea black tussock moth (D. baibarana) based on the FPKM values. (A) P450s with higher FPKM values in the pheromone gland; (B) P450s with higher FPKM values in the abdomen. Pg and Ab indicated the pheromone gland and the abdomen, respectively. Asterisks represent significant differences between the two tissues based on the Bonferroni t-test (* p < 0.05, *** p < 0.001).

Figure 3

Phylogenetic analysis of D. baibarana CYPs. The protein sequences of DbCYPs with more than 100 amino acids were used for tree construction using neighbor-joining method. The bootstrap replicates were 1000. Numbers at each branch point represent the bootstrap values. Different background colors indicated CYP2 clan (brow), CYP3 clan (purple), CYP4 clan (green), and CYP mitochondrial clan (light blue).

Figure 4

Phylogenetic analysis of D. baibarana CYPs expressed predominantly in pheromone glands with those from other insect species. The tree was conducted with MEGA 7.0 based on the amino acid sequences using the maximum likelihood method based on the Poisson model. The bootstrap replicates were 1000. Numbers at each branch point represent the bootstrap values. Different background colors indicated CYP3 clan (brown), CYP4 clan (light blue), and CYP mitochondrial clan (purple). Species abbreviations are as follows: Db, Dasychira baibarana; Sl, Spodoptera litura; Sf, Spodoptera frugiperda; Ha, Helicoverpa armigera; Se, Spodoptera exigua; Hc, Hyphantria cunea; Ms, Manduca sexta; Bm, Bombyx mori; Ob, Operophtera brumata; Hz, Helicoverpa zea; Sli, Spodoptera littoralis; Mb, Mamestra brassicae; Of, Ostrinia furnacalis; Cs, Chilo suppressalis; Cm, Cnaphalocrocis medinalis; Ld, Lymantria dispar; Hv, Heortia vitessoides; Li, Lemyra imparilis; As, Ascotis selenaria.

Figure 5

Tissue expressional profiles of the 2 CYP4Gs and 17 pheromone-gland-enriched CYPs in D. baibarana based on qPCR analysis. (A) expression level of the two CYP4Gs; (B) the nine pheromone-gland up-regulated expressed CYPs; (C) the eight antennae up-regulated expressed CYPs. Abbreviations: Pg, pheromone gland; Ab, abdomen; An, antennae; H, head; L, leg; T, thorax. Different letters indicated the expression levels are significantly different (p < 0.05).