Effect of Silencing subolesin and enolase impairs gene expression, engorgement and reproduction in Haemaphysalis longicornis (Acari: Ixodidae) ticks

Abstract

Importance: Haemaphysalis longicornis is an obligate blood-sucking ectoparasite that has gained attention due its role of transmitting medically and veterinary significant pathogens and it is the most common tick species in Republic of Korea. The preferred strategy for controlling ticks is a multi-antigenic vaccination. Testing the efficiency of a combination antigen is a promising method for creating a tick vaccine.

Objective: The aim of the current research was to analyze the role of subolesin and enolase in feeding and reproduction of H. longicornis by gene silencing.

Methods: In this study, we used RNA interference to silence salivary enolase and subolesin in H. longicornis. Unfed female ticks injected with double-stranded RNA targeting subolesin and enolase were attached and fed normally on the rabbit's ear. Real-time polymerase chain reaction was used to confirm the extent of knockdown.

Results: Ticks in the subolesin or enolase dsRNA groups showed knockdown rates of 80% and 60% respectively. Ticks in the combination dsRNA (subolesin and enolase) group showed an 80% knockdown. Knockdown of subolesin and enolase resulted in significant depletion in feeding, blood engorgement weight, attachment rate, and egg laying. Silencing of both resulted in a significant (p < 0.05) reduction in tick engorgement, egg laying, egg hatching (15%), and reproduction.

Conclusions and relevance: Our results suggest that subolesin and enolase are an exciting target for future tick control strategies.

Keywords: RNA interference; Real-time PCR; double-stranded RNA; knockdown; transcript; vaccine.

Fig. 1. Real-time polymerase chain reaction results indicated genes in the group treated with subolesin + enolase combinly shows significant decrease expression level following RNA interference than that of the gene subolesin and enolase separately treated with a single group. The data represent mean ± SD, normalized to the actin transcript level.

^*p < 0.05, compared with the control group as determined by a Student’s t-test.

Fig. 2. Effects of subolesin and enolase dsRNA on tick engorgement and reproduction; (A) Group treated with Subolesin and enolase dsRNA. Significantly different compared to the control group (injection buffer); (B) Average engorgement weight in the group treated with Subolesin and Enolase dsRNA. Significantly different compared to the control group as determined by a Student’s t-test; (C) Effect of Subolesin and Enolase dsRNA on tick attachment, highly significant compared to the control group; (D) Death rate of Haemaphysalis longicornis adult ticks 24 h after injection of Subolesin and Enolase dsRNA. Significantly different compared to the control group; Data are presented as median ± SD.

dsRNA, double stranded RNA. ^***p < 0.001; ^****p < 0.0001.

Fig. 3. Phenotypic changes of ticks after infestation in rabbit ear with dsRNA (subolesin + enolase) compared to control (injection buffer) injection. Changes in tick morphology during feeding several days after injecting subolesin + enolase dsRNA (A) 4^th day; (B) 6^th day; (C) 8^th day; (D) 10^th day; Compare to control group (Injection buffer); (E) 4^th day; (F) 6^th day; (G) 8^th day.

dsRNA, double stranded RNA.

Fig. 4. Phenotypic changes in tick (Haemaphysalis longicornis) engorgement and egg morphology after subolesin + enolase dsRNA treatment. (A) Control ticks (Injection buffer); (B) Subolesin+ enolase dsRNA-treated ticks after spontaneous drop-down; (C) Control tick eggs; (D) Abnormal eggs of subolesin + enolase dsRNA-treated ticks; (E) Control tick eggs containing embryos; (F) Subolesin+ enolase dsRNA-treated ticks laid abnormal eggs (some have no embryos). Scale bar = (C-F) 10 μm.

dsRNA, double stranded RNA.

Fig. 5. Effects of subolesin+ enolase dsRNA on tick engorgement and reproduction. (A) Average egg mass weight in the treated group and the control group (injection buffer); (B) Average egg conversion ratio; (C) Average egg hatching ratio among treated group compared with the control group as determined by a Student’s t-test. Data are presented as median ± SD.

dsRNA, double stranded RNA. ^****p < 0.0001.