Functional characterization of a tyrosine hydroxylase in Diaphorina citri and utilization of star polycation nanoparticle-wrapped dsRNA for pest control

Abstract

Background: The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae), a notorious pest accountable for citrus Huanglongbing (HLB), has developed high levels of resistance to a variety of commercial insecticides. RNA interference (RNAi) is a promising and effective approach for pest management. The first step for effective pest control by RNAi is the development of effective and reliable target genes. Tyrosine hydroxylase (TH) plays important roles in immunity, cuticle tanning and pigmentation.

Results: In this study, we focused on the functions of TH during the molting of D. citri, and whether TH could be employed as a target for the pest control. The D. citri tyrosine hydroxylase (DcTH) complementary DNA (cDNA) sequence was identified from the genome database of D. citri and cloned. Its open reading frame (ORF) was 1782 bp, encoding a protein consisting of 593 amino acids. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis demonstrated that DcTH was highly expressed in the wing, and its expression level was significantly higher in the early stages of each instar compared with the late stages. Additionally, the expression level of DcTH was higher in the early stage of molting than in the late stage, while TH activity presented the opposite trend. The 3-iodo-tyrosine (3-IT) treatment or double-stranded RNA (dsRNA) injection significantly decreased the expression level of DcTH and TH activity, and augmented the mortality of D. citri. The silencing of DcTH by RNAi significantly disrupted the metamorphosis to the adult stage and reduced the content of 3,4-dihydroxyphenylalanine (DOPA) in D. citri. Transmission electron microscopy (TEM) analysis indicated that the synthesis of the exocuticle was suppressed following the inhibition of DcTH. Furthermore, the down-regulation of DcTH significantly inhibited the expression levels of three cuticle tanning pathway genes. Additionally, a drop RNAi method targeting DcTH based on star polycation (SPc) nanoparticles-wrapped dsRNA significantly increased the mortality of D. citri.

Conclusions: These results imply that DcTH plays a crucial role in exocuticle tanning in D. citri, and it could be an ideal target for RNAi-mediated control of D. citri. © 2025 Society of Chemical Industry.

Keywords: Diaphorina citri; RNA interference; cuticle formation; star polycation; tyrosine hydroxylase.