Localized expression and inhibition effect of miR-184 on blood digestion and oviposition in Haemaphysalis longicornis (Acari: Ixodidae)

Abstract

Background: The hard tick Haemaphysalis longicornis (Ixodidae) is widely distributed in East Asia, China, Australia and New Zealand. It can transmit many infectious pathogens, including the causative agents of human rickettsiosis, bovine theileriosis, bovine babesiosis and canine babesiosis. Therefore, a greater understanding of H. longicornis biology might aid in the development of more effective control measures against the tick and tick-borne pathogens.

Methods: We analyzed the expression of miR-184 in different developmental stages and various tissues of H. longicornis using real-time PCR (qRT-PCR). Antagomir (Ant-184) was used to knock-down miR-184, whilst Ms-Ant and non-injected ticks were used as the negative and blank controls, respectively. We used online software tools (RNAhybrid and TargetScan) to predict the putative target genes of miR-184.

Results: The expression of miR-184 was highest in unfed nymphs and lowest in unfed larvae. The tissue distribution of miR-184 showed abundant expression in the midgut. To investigate the probable roles of miR-184, antagomir (Ant-184) was used to knock-down miR-184 (t₍₄₎ = 12.32, P = 0.0002). After inhibiting miR-184, other biological factors were examined in each group. The engorged body weight was significantly reduced in the treated group (Ant-184) in contrast to control groups (t₍₂₂₎ = 2.19, P = 0.0388). The mean duration of the egg-laying days was significantly increased (33.5 ± 1.91) and the number of eggs (t₍₁₀₎ = 3.147, P = 0.0137), and egg mass (t₍₁₀₎ = 3.4472, P = 0.0063) were significantly reduced in the treated group. During oviposition, eggs were monitored and in half of the ticks of the Ant-184 group the eggs were completely desiccated, lacked embryo development and did not hatch. We analyzed the expression of Vg proteins (Vg1, Vg2, Vg3) in semi-engorged ticks, engorged ticks, ticks at day 2 after engorgement and egg stage in Ant-184, non-injected and Ms-Ant groups, and found significant variation.

Conclusions: This study provides information on the role of miR-184 in H. longicornis ticks. The data suggest that miR-184 targets Vg proteins and affects blood digestion and oviposition.

Keywords: Blood digestion; Haemaphysalis longicornis; Oviposition; Vitellogenin; miR-184.

Fig. 1

miR-184 expression in different developmental stages and tissues. a Relative expression of miR-184 was analyzed in eggs, unfed larvae, fed larvae, unfed nymphs, fed nymphs, unfed adults and fed adults. b Relative expression of miR-184 in midgut, salivary gland and ovary in unfed and fed female ticks. Data represent three biological replicates with three technical replicates and are shown as the mean ± SEM

Fig. 2

Relative miR-184 expression in the fed adult ticks. The miR-184 inhibited by Antagomir-184. Data represent three biological replicates with three technical replicates and are shown as the mean ± SEM

Fig. 3

Average engorged weight after silencing in treated and control groups. Data represent three biological replicates with three technical replicates and are shown as the mean ± SEM

Fig. 4

Silencing of miR-184 affects blood digestion. Average numbers of eggs (a) and egg mass (b) in non-injected ticks, Ms-Ant and Ant-184 ticks. The data are shown as the mean ± SEM

Fig. 5

miR-184 inhibition affects egg development. Non-injected (a) and Ms-Ant control (c) showed normal embryo development, whereas in the Ant-184-treated group (b) eggs became desiccated and lacked development. Scale-bars: 1000 µm

Fig. 6

Relative expression of Vg proteins in semi-engorged ticks. Expression of Vg1 (a), Vg2 (b) and Vg3 (c) in treated (Ant-184) and control groups. Data represent three biological replicates with three technical replicates and are shown as the mean ± SEM

Fig. 7

Relative expression of Vg proteins in engorged ticks. Expression of Vg1 (a), Vg2 (b) and Vg3 (c) in non-injected, Ms-Ant and Ant-184 groups. Data represent three biological replicates with three technical replicates and are shown as the mean ± SEM

Fig. 8

Relative expression of Vg proteins at day 2 after engorgement. Expression of Vg1 (a), Vg2 (b) and Vg3 (c) in non-injected, Ms-Ant and Ant-184 groups. Data represent three biological replicates with three technical replicates and are shown as the mean ± SEM

Fig. 9

Relative expression of Vg proteins at egg stage after inhibition. Expression of Vg1 (a),Vg2 (b) and Vg3 (c) in non-injected, Ms-Ant and Ant-184 groups. Data represent three biological replicates with three technical replicates and are shown as the mean ± SEM