Theileria and Babesia protozoan parasites are transmitted mainly by tick vectors. These parasites cause heavy economic losses to the live-stock industry, as well as affecting the health of wild animals in parasite-endemic areas. Identification of infectious agents in wild animals is not only crucial for species preservation, but also provides valuable information on parasite epidemiology. Here, we conducted a molecular surveillance study in Northwestern China to assess the prevalence of blood pathogens in cervids.

The hard tick Haemaphysalis longicornis is widely distributed in eastern Asia, New Zealand and Australia and is considered the major vector of Theileria and Babesia, harmful parasites to humans and animals. Female ticks need successful blood meals to complete the life-cycle. Therefore, elucidation of the underlying molecular mechanisms of H. longicornis development and reproduction is considered important for developing control strategies against the tick and tick-borne pathogens. Luciferase assays were used to identify the targets of micro RNA miR-275 in vitro. RNAi of Vitellogenin (Vg) was used in phenotype rescue experiments of ticks with miR-275 inhibition, and these analyses were used to identify the authentic target of miR-275 in vivo. The expression of miR-275 in different tissues and developmental stages of ticks was assessed by real-time PCR. To elucidate the functions of miR-275 in female ticks, we injected a miR-275 antagomir into female ticks and observed the phenotypic changes. Statistical analyses were performed with GraphPad5 using Student’s t-test. In this study, we identified Vg-2 as an authentic target of miR-275 both in vitro and in vivo by luciferase assays and phenotype rescue experiments. miR-275 plays the regulatory role in a tissue-specific manner and differentially in developmental stages. Silencing of miR-275 resulted in blood digestion problems, substantially impaired ovary development and significantly reduced egg mass (P < 0.0001). Furthermore, RNAi silencing of Vg-2 not only impacted the blood meal uptake (P < 0.05) but also the egg mass (P < 0.05). Significant rescue was observed in miR-275 knockout ticks when RNAi was applied to Vg-2. To our knowledge, this study is the first demonstration that miR-275 targets Vg-2 in H. longicornis and regulates the functions of blood digestion and ovary development. These findings improve the molecular understanding of tick development and reproduction.

Theileria and Anaplasma are especially important emerging tick-borne pathogens of animals and humans. Molecular surveys and identification of the infectious agents in Mongolian gazelle, Procapra gutturosa are not only crucial for the species’ preservation, but also provide valuable information on parasite and bacterial epidemiology. A molecular surveillance study was undertaken to assess the prevalence of Theileria spp. and Anaplasma spp. in P. gutturosa by PCR in China. Theileria luwenshuni, A. bovis, A. phagocytophilum, and A. ovis were frequently found in P. gutturosa in China, at a prevalence of 97.8%, 78.3%, 65.2%, and 52.2%, respectively. The prevalence of each pathogens in the tick Haemaphysalis longicornis was 80.0%, 66.7%, 76.7%, and 0%, respectively, and in the tick Dermacentor niveus was 88.2%, 35.3%, 88.2%, and 58.5%, respectively. No other Theileria or Anaplasma species was found in these samples. Rickettsia raoultii was detected for the first time in P. gutturosa in China. Our results extend our understanding of the epidemiology of theileriosis and anaplasmosis in P. gutturosa, and will facilitate the implementation of measures to control these tick-borne diseases in China.

Ticks are blood-sucking arthropods that can transmit diseases to humans and animals. These arthropods are the second most important vectors of pathogens. MicroRNAs are a class of conserved small noncoding RNAs that play regulatory roles in gene expression at the post-transcriptional level. Molting is an important biological process in arthropods. Research on the molting process is important for understanding tick physiology and control. Dual-luciferase reporter assays were used to assess the role of miRNA let-7 in ecdysteroid receptor (ECR) biology. The expression levels of ECR and let-7 were measured by real-time qPCR before and after tick molting. To explore the function of let-7 and ECR, we performed overexpression and knocking down of let-7 and RNAi of ECR in tick nymphs. The biological function of let-7 in molting was explored by injecting nymphs, ten days after engorgement, with let-7 agomir for overexpression and let-7 antagomir for knocking down. The rate of molting was then determined. ECR dsRNA was injected into ticks to evaluate the function of ECR by gene silencing. The expression of ECR and let-7 was measured using RT-qPCR. All data were analyzed using GraphPad Prism v.6. The results of the luciferase assay using a eukaryotic expression system revealed that ECR was a natural target of let-7. Let-7 overexpressed by agomir affected the rate of molting (P < 0.01) and the period of molting (P < 0.01). Let-7 antagomir for knockdown affected the period of molting (P < 0.01), but there was no effect on the rate of molting (P = 0.27). ECR dsRNA gene silencing significantly affected the rate of molting (P < 0.05). This study demonstrated that let-7 can regulate the expression of ECR and that let-7 can affect molting in ticks. Our results help to understand the regulation of let-7 by 20-hydroxyecdysone (20E) and will provide a reference for functional analysis studies of microRNAs in ticks.