Characterization of Esterase Genes Involving Malathion Detoxification and Establishment of an RNA Interference Method in Liposcelis bostrychophila

Abstract

Esterases (ESTs) play important roles in metabolizing various physiologically endogenous and exogenous compounds, and various environmental xenobiotics in insects. The psocid, Liposcelis bostrychophila is a major pest of stored products worldwide and rapidly develops resistance to commonly insecticides. However, the involvement of ESTs in insecticide metabolization and the application of RNAi approach in psocids have not been well elucidated. In this study, we characterized four LbEST genes and investigated the transcriptional levels of these genes at different developmental stages and under different insecticides exposures to assess their potential roles in response to insecticides. The four LbESTs contain a catalytic triad (Ser-His-Glu) linked to an oxyanion hole and acyl pocket involved in substrate stabilization during its hydrolysis. Synergism observed with the esterase-inhibitor DEF suggests the involvement of esterases in malathion detoxification. LbESTs were expressed during the whole of developmental stages, but predominant abundance in the first nymphal instar and adult stage. The mRNA level of three LbEST genes (except for LbEST4) was induced (1.29- to 5.60 fold) in response to malathion or deltamethrin exposures, indicating that these esterases are involved in the detoxification process. Silencing of LbEST1, LbEST2 or LbEST3 through dsRNA feeding led to a higher mortality of psocids upon the malathion treatment compared to controls (1.83 to 2.69-fold), demonstrating that these esterase genes play roles in malathion detoxification in L. bostrychophila. Our study provides new evidence for understanding of the function and regulation mechanism of esterases in L. bostrychophila in insecticide detoxification. The current study also suggests that the present RNAi method could be applied for gene functional studies in psocids.

Keywords: RNAi; booklice; detoxification enzyme; microsatellite; stored product pests.

FIGURE 1

Workflow of Gene knock-down by RNA interference in psocids.

FIGURE 2

Alignment of the deduced amino acid sequence of four Liposcelis bostrychophila esterase genes. The signal peptide is underlined with a solid line in bold letters; the conserved motifs are in green shadow; the catalytic triads are marked with black box; the residues for the oxyanion hole are linked with red box; the esterase conserved motif, GxSxG, is marked with blue letters and underlined with a blue solid line; The type-B carboxylesterase signature 2 motif and serine motif are highlighted in yellow (red letters) and gray (blue letters), respectively.

FIGURE 3

An un-rooted Neighbor-joining tree constructed based on the esterase amino acid sequences from Liposcelis bostrychophila and other insects. Esterase genes from L. bostrychophila (Lb), Musca domestica (Md), and Drosophila melanogaster (Dm) are marked by filled rhombus (red color indicates for esterases; brown color indicates for acetylcholinesterases), blue filled circle and black filled triangle, respectively. Different clades are shown in different colors. Nodes with >50% bootstrap values are shown. All insect esterase sequences were retrieved from NCBI.

FIGURE 4

Relative expression levels of LbESTs determined by qPCR in different developmental stages. Stage-dependent expression patterns including egg, first, second, third, and fourth-instar nymph, and adults. Bar graph presents values as mean ± SE. Different letters above the error bar for each gene show significant difference (P < 0.05).

FIGURE 5

Time course effects of two different insecticides exposure (LC₅₀) on the expression profiles of four esterase genes in Liposcelis bostrychophila. Bar graph presents values as mean ± SE. Asterisks (*) above the error bars indicate statistical differences determined by the independent samples t-test (*P < 0.05; **P < 0.01; ***P < 0.001).

FIGURE 6

The relative expression of four LbEST genes was determined in Liposcelis bostrychophila by feeding their corresponding dsRNA. (A) The relative expression of four esterase genes in L. bostrychophila after feeding their dsRNA separately for 24 h; (B) The relative expression of four esterase genes in L. bostrychophila after feeding their dsRNA separately for 48 h; GFP-dsRNA was used as the control; Asterisks (*) above the error bars indicate statistical differences determined by the independent samples t-test (*P < 0.05; **P < 0.01).