Multiple Glutathione S-Transferase Genes in Heortia vitessoides (Lepidoptera: Crambidae): Identification and Expression Patterns

Abstract

To elucidate the role of glutathione S-transferases (GSTs) in Heortia vitessoides Moore (Lepidoptera: Crambidae), one of the most destructive defoliating pests in Aquilaria sinensis (Lour.) Gilg (Thymelaeaceae) forests, 16 GST cDNAs were identified in the transcriptome of adult H. vitessoides. All cDNAs included a complete open reading frame and were designated HvGSTd1-HvGSTu2. A phylogenetic analysis showed that the 16 HvGSTs were classified into seven different cytosolic classes; three in delta, two in epsilon, three in omega, three in sigma, one in theta, two in zeta, and two in unclassified. The expression patterns of these HvGSTs in various larval and adult tissues, following exposure to half the lethal concentrations (LC50s) of chlorantraniliprole and beta-cypermethrin, were determined using real-time quantitative polymerase chain reaction (RT-qPCR). The expression levels of the 16 HvGSTs were found to differ among various larval and adult tissues. Furthermore, the RT-qPCR confirmed that the transcription levels of nine (HvGSTd1, HvGSTd3, HvGSTe2, HvGSTe3, HvGSTo3, HvGSTs1, HvGSTs3, HvGSTu1, and HvGSTu2) and six (HvGSTd1, HvGSTd3, HvGSTe2, HvGSTo2, HvGSTs1, and HvGSTu1) HvGST genes were significantly higher in the fourth-instar larvae following exposure to the insecticides chlorantraniliprole and beta-cypermethrin, respectively. These genes are potential candidates involved in the detoxification of these two insecticides. Further studies utilizing the RNA interference approach are required to enhance our understanding of the functions of these genes in this forest pest.

Fig. 1.

Sequence alignment of the conserved GST sites from Bombyx mori (Bm), Pieris rapae (Pr), Cnaphalocrocis medinalis (Cm), Chilo suppressalis (Cs), and Heortia vitessoides (Hv). The amino acid residues not shown here are represented by three sequential dots. The predicted GSH-binding sites (G-sites) are shaded in light gray and the substrate-binding pockets (H-sites) are shaded in dark gray. The GenBank accession numbers are shown in Supp Table 1 [online only].

Fig. 2.

Phylogenetic analysis of GSTs from Pieris rapae (Pr), Bombyx mori (Bm), Cnaphalocrocis medinalis (Cm), Chilo suppressalis (Cs), Spodoptera litura (Sl), and Heortia vitessoides (Hv). Insect GSTs are classified into seven distinct classes (delta, epsilon, omega, sigma, theta, zeta, and unclassified). The 16 H. vitessoides GSTs (HvGSTs) are highlighted with black circles. The GenBank accession numbers and amino acid sequences used for phylogenetic tree construction are given in Supp Table 1 [online only].

Fig. 3.

Relative expression levels of 16 HvGST genes in various larval tissues. L (2-d-old fourth-instar larvae), MG (midgut), MT (Malpighian tubules), and FB (fat body). The HvGST expression levels in different larval tissues were normalized relative to those in the 2-d-old fourth-instar larvae. Different letters indicate significant differences at P < 0.05 according to one-way analysis of variance (ANOVA), followed by Tukey’s test. The data represent the mean ± SD of three biological replicates.

Fig. 4.

Relative expression levels of 16 HvGST genes in various adult tissues. A (3-d-old adults), AN (antenna), AB (abdomen), and LE (leg). The HvGST expression levels in different adult tissues were normalized relative to those in the 3-d-old adults. Different letters indicate significant differences at P < 0.05 according to one-way analysis of variance (ANOVA), followed by Tukey’s test. The data represent the mean ± SD of three biological replicates.

Fig. 5.

Relative expression levels of 16 HvGST genes in the fourth-instar larvae exposed to the half lethal concentrations (LC₅₀s) of chlorantraniliprole and beta-cypermethrin. The dashed line represents the normalized level of gene expression in the control larvae. The asterisk (*) indicates a significant difference in transcription levels between the treated and control insects (paired Student’s t-test, P < 0.05). The data represent the mean ± SD of three biological replicates.