Molecular cloning, identification, transcriptional analysis, and silencing of enolase on the life cycle of Haemaphysalis longicornis (Acari, Ixodidae) tick

Abstract

Ticks, blood-sucking ectoparasites, spread diseases to humans and animals. Haemaphysalis longicornis is a significant vector for tick-borne diseases in medical and veterinary contexts. Identifying protective antigens in H. longicornis for an anti-tick vaccine is a key tick control strategy. Enolase, a multifunctional protein, significantly converts D-2-phosphoglycerate and phosphoenolpyruvate in glycolysis and gluconeogenesis in cell cytoplasm. This study cloned a complete open reading frame (ORF) of enolase from the H. longicornis tick and characterized its transcriptional and silencing effect. We amplified the full-length cDNA of the enolase gene using rapid amplification of cDNA ends. The complete cDNA, with an ORF of 1,297 nucleotides, encoded a 432-amino acid polypeptide. Enolase of the Jeju strain H. longicornis exhibited the highest sequence similarity with H. flava (98%), followed by Dermacentor silvarum (82%). The enolase motifs identified included N-terminal and C-terminal regions, magnesium binding sites, and several phosphorylation sites. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that enolase mRNA transcripts were expressed across all developmental stages of ticks and organs such as salivary gland and midgut. RT-PCR showed higher transcript levels in syn-ganglia, suggesting that synganglion nerves influence enolase,s role in tick salivary glands. We injected enolase double-stranded RNA into adult unfed female ticks, after which they were subsequently fed with normal unfed males until they spontaneously dropped off. RNA interference significantly (P<0.05) reduced feeding and reproduction, along with abnormalities in eggs (no embryos) and hatching. These findings suggest enolase is a promising target for future tick control strategies.

Keywords: Double-stranded RNA; RNA interference; knockdown; real-time PCR; transcript; vaccine.

Fig. 1

Multiple alignment of enolase amino acid sequence in ticks. The alignment of enolase amino acid sequence from Dermacentor silvarum (XM_037726356.1), Ixodes scapularis (XM_029979980.4), Rhipicephalus microplus (MW678616.1), Amblyomma parvum (GBBL01001179.1), and Haemaphysalis flava (KM191327.1). Asterisks indicate conserved residues. Motifs are indicated by boxed letters.

Fig. 2

Phylogenetic analysis of enolase from H. longicornis. Bootstrap proportions are indicated at branches. Sequences with NJ involving Poisson corrections and bootstrap analysis of 500 replicates. H. longicornis and R. flava are in the same clade.

Fig. 3

Transcriptional profiles of enolase (A) knockdown of enolase compared with the control group; (B) enolase expression in fed and unfed midguts of adult females. **P<0.001 by Student’s t-test.

Fig. 4

Transcriptional profiles of enolase at different developmental stages (A) enolase expression in unfed versus fed adult ticks. (B) Enolase expression in salivary glands of unfed versus fed adult ticks. **P<0.01, ***P<0.001 by Student’s t-test.

Fig. 5

Transcriptional profiles of enolase expression in adult, nymph, larvae, and egg. *P<0.05 by Student’s t-test.

Fig. 6

Phenotypic changes in tick (Haemaphysalis longicornis) egg morphology and reduce egg hatching ability compared with. 1, non-injected; 2, buffer control; 3, enolase dsRNA-treated group; (A, B) control ticks, eggs and Control tick eggs containing embryos; (C) enolase dsRNA-treated ticks after spontaneous drop-down, eggs, Abnormal eggs (no embryos).

Fig. 7

Morphological changes of adult H. longicornis after RNAi silencing of enolase. Upper panel shows RHAi induced enolase knockdown ticks and lower panel represents control ticks.