MicroRNA-275 and its target Vitellogenin-2 are crucial in ovary development and blood digestion of Haemaphysalis longicornis
© The Author(s). 2017
Received: 13 March 2017
Accepted: 21 April 2017
Published: 22 May 2017
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.
KeywordsHaemaphysalis longicornis MicroRNA Vitellogenin Blood digestion Ovary development
Ticks are hematophagous ectoparasites that require blood meals from vertebrates to obtain a variety of nutrients. They transmit many vector-borne pathogens to humans and animals  and cause significant economic losses [2, 3]. Ticks are considered one the most common vectors of human and animal diseases worldwide, second only to mosquitoes . The hard tick Haemaphysalis longicornis is found in eastern Asia, New Zealand and Australia and is distributed in most regions of China. This species carries Theileria spp. and Babesia spp., which are harmful parasites in humans and animals [5, 6].
MicroRNAs (miRNAs) are the most abundant class of small non-coding RNAs. They are 19–24 nt in length and regulate gene expressions at the post-transcriptional level by binding to target mRNAs [7, 8]. In animals, miRNAs recognize their target sites by incomplete base pairing , and nucleotides 2–7 (the seed region) are complementary to the target mRNA. Most miRNAs act on 3' untranslated regions (3' UTR) of the target mRNA , but they can also bind to the 5' UTR [11, 12] and the open reading frame (ORF) [13, 14].
Vitellogenin (Vg), common to oviparous animals, is a class of phospho-lipoglycoprotein and is the precursor of Vitellin . In ticks, Vg is predominantly synthesized in the fat body and regulated by 20-hydroxyecdysone; it is transported to specific tissues after a blood meal [16, 17]. Vg provides amino acids, carbohydrates and other nutrients for embryonic development. Vg is not only found in fed adults but also in larvae and eggs . Multiple Vgs were previously detected in different tissues of H. longicornis . Vg could regulate the expression of microRNAs and may affect foraging behavior in honey bees . In zebrafish, miRNA expression was also associated with Vgs, and six differentially expressed miRNAs may potentially interact with Vgs . Although several tick specific-miRNAs have been identified in different developmental stages , thus far, in vivo functional studies are lacking. A previous study demonstrated that miRNA-275 is crucial for blood digestion and egg development in Aedes aegypti . miR-275 is one of the 10 most abundant miRNAs in female Rhipicephalus haemaphysaloides ticks, but not in males, indicating that miR-275 has a critical role in females . However, the function of miRNA-275 in ticks is unclear. Thus, to determine the roles of miRNA-275 and Vg in H. longicornis ticks, we identified the putative miR-275 binding sites within Vg-2 and examined the role of miR-275 in regulating ovarian development and blood digestion.
Haemaphysalis longicornis ticks were collected from the Gansu Province and have been maintained in our laboratory since 2006. Adult ticks fed on New Zealand white rabbits. After detachment, the ticks were reared at 28 ± 1 °C and 75% humidity in a drying basin.
Computational prediction of miRNA targets
To predict putative gene targets of miR-275 (MI0012273), we used the RNAhybrid programme to identify miR-275 binding sites in the complete sequence of Vgs. There are four known H. longicornis Vgs (HlVg-1, HlVg-2, HlVg-3 and HlVg-B) available in GenBank-NCBI, and we selected HlVg-1, HlVg-2, and HlVg-3 for analysis because the specific functions of HlVg-B are unknown.
Cell culture and luciferase assay
The 293 T cell line was used in this study. Cells were maintained in Dulbecco's modified Eagle’s medium (DMEM) (Gibco, Waltham, USA) supplemented with 10% fetal bovine serum (Gibco), penicillin and streptomycin and maintained in an incubator with 5% CO2 at 37 °C. The predicted binding sites were cloned and then inserted into the pmirGLO (Promega, Madison, USA) reporter vector. For reporter assays, 150 ng of pmirGLO reporter vector and 50 nM miR-275 mimic (RiboBio, GuangZhou, China) were co-transfected into 293 T cells using Lipofectamine 2000. No-mimic treatment cells were used as a blank control, and pmirGLO-Vg vector only cells were used as the negative control. Firefly and Renilla luciferase activities were measured 48 h post-transfection by the Dual-Luciferase Reporter Assay System (Promega). First, 100 μl of luciferase assay reagent II was added to each well, Firefly luciferase activities was measured, and 100 μl Stop&Glo reagent was loaded into each well. Then, Renilla luciferase activities were measured. Firefly luciferase in the pmirGLO vector was used for normalization of Renilla luciferase expression. Treatments were assessed in triplicate, and transfections were repeated three times. Firefly luciferase activities were divided by Renilla luciferase activities. Finally, the ratio between Renilla and Firefly luciferase activities was calculated for each experiment.
Synthesis and application of antagomir
Antagomirs are miRNA-specific antisense oligonucleotides and were synthetized by Dharmacon (http://dharmacon.gelifesciences.com). The miRNA-275 antagomir (Ant-275) was the reverse complement of mature miRNA-275, and chemical modification was performed as described in a previous study  (5'-mC.*.mG.*.mC.mG.mC.mG.mC.mU.mA.mC.mU.mU.mC.mA.mG.mG.mU.mA.mC.mC.*.mU.*.mG.*.mA.*-Chl-3'). The “missense” (MsAnt) sequence (5'-mC.*.mG.*.mC.mU.mU.mU.mC.mG.mU.mG.mG.mU.mU.mC.mU.mG.mG.mU.mA.mC.*.mC.*.mU.*.mU.*-Chl-3') was used as the negative control of the antagomir. [“*” is a phosphate backbone modification that was introduced to increase nuclease resistance and facilitate cellular uptake and bioavailability in vivo. “m” is a 2'-O-methyl (2'-OMe) modification, which reduces off-targeting. “Chl” is cholesterol, which can enhance gene silencing in vivo]. Non-injected ticks were used as a blank control group. To assess the specificity of the antagomir, we measured the levels of other miRNAs, such as miRNA-10. Antagomirs were microinjected into unfed adult female H. longicornis at a dose of 400 μM in 0.5 μl. Every group had 30 female ticks in total, and the groups fed on animals for blood-feeding 24 h after microinjection.
Phenotype rescue experiment and Vg-2 RNAi
Phenotype rescue experiment group
No of ticks
400 μM + 2 μg
400 μM + 2 μg
Total RNA isolation and real-time PCR
Total RNA was extracted from various tissues and development stages of ticks with TRIzol reagent (TaKaRa, Dalian, China) according to the manufacturer’s protocol, and miScript (TaKaRa) was utilized for mRNA and miRNA cDNA synthesis. Real-time PCR was performed to analyze the expression of miRNA and mRNA using a SYBR Prime Script miRNA RT-PCR kit. Specific primers for Vg-2 (forward primer 5'-TGG TGG ACA AGG GAT GAG AA-3' and reverse primer 5'-ATC CGA GCG AAC TGA AGA CC-3'), miRNA-275 (5'-CAG GTA CCT GAA GTA GCG CG-3') and miRNA-10 (5'-ACC CTG TAG ATC CGA ATT TGT-3') were used in this study. U6 and β-actin were also amplified as internal controls. Relative expression of miRNAs and Vg-2 was calculated by the 2-ΔCt method.
All data were analyzed with GraphPad 5 using Student’s t-test. Probability values of less than 0.05 were considered significant, and the results are shown as the mean ± SEM.
miR-275 targets Vg-2 in vitro
Expression analysis of miR-275 and Vg-2 from various developmental stages and tissues in ticks
Inhibition of miR-275 severely affects blood digestion and ovary development
The effect of Ant-275 on blood digestion of Haemaphysalis longicornis. Data are shown as the mean ± SEM
Change rate of engorged weight (%)a
Duration of laying eggs (days)
Final weight (mg)b
69.33 ± 1.547*
28.10 ± 0.246*
66.40 ± 1.785***
82.69 ± 0.943
26.30 ± 0.329
37.00 ± 0.875
83.94 ± 0.913
25.33 ± 0.226
35.23 ± 1.003
Phenotype rescue experiment
Effect of Vg-2 dsRNA treatment. Data are shown as the mean ± SEM
Engorged weight (mg)
Egg weight/Body weight (%)
98.77 ± 2.268***
25.9 ± 0.9572***
198.68 ± 6.880
54.1 ± 1.559
211.20 ± 3.593
58.6 ± 1.986
Vgs have many miR-275 binding sites, but only Vg-2 had two binding sites with a strong 7-mer seed match site at positions 2–8 for miR-275. A previous study showed that Vg-2 was detected in the ovary and is crucial for ovarian development; therefore, we chose these two canonical seed binding sites for further study. A luciferase reporter vector co-transfected with miR-275 mimic drastically inhibited the Renilla luciferase activity, which demonstrated that Vg-2 is a miR-275 target in vitro. Furthermore, Vg-2 RNAi with Ant-275 partially alleviated egg deposition and blood-feeding and has been successfully used in rescuing miRNA mutant phenotypes in mosquitoes and Drosophila [24, 25]. Thus, the Dual-Luciferase reporter assay system and RNAi demonstrated in vitro and in vivo that Vg-2 is an authentic target of miR-275 in H. longicornis. This is the first report to use the Dual-Luciferase reporter system to verify microRNAs and their target genes in ticks.
Shiping Liu et al.  showed that injection of Ant-1174 significantly decreased the egg deposition, but co-injection of Ant-1174/dsSHMT partially reduced the egg deposition, which demonstrated that SHMT (serine hydroxymethyltransferase) was a target gene of miRNA-1174 in vivo. In this study, co-injection of dsVg and Ant-275 partially recovered the phenotype; miR-275 was inhibited and did not bind to Vg, and therefore, Vg was only inhibited by dsRNA. These results are similar to those of a previous study.
In a previous study, miR-275 was shown to play an important role in egg development and blood digestion in the mosquito Aedes aegypti . Similarly, our study was first to demonstrate that miR-275 and its target gene Vg-2 also regulated blood digestion and ovary development in ticks. Inhibition of H. longicornis miR-275 weakened the capability for blood digestion and had severe effects on ovarian development and egg deposition. Ovaries were substantially smaller after inhibition of miR-275, which implied that decreased egg deposition may be attributed to the failed development of the ovaries. Similar to a previous study , we found that the egg number and the engorged weight were significantly reduced with Vg-2 RNAi. Because the expression of Vg was very low in unfed ticks (increased after feeding) and barely detected by real-time PCR, we assessed the efficiency of Vg-2 RNAi after feeding and found that it was 51.4%, which satisfied the experimental requirements.
Expression analysis of miR-275 and Vg-2 in different developmental stage of ticks showed that miR-275 and Vg-2 increased in the fed adult stage, suggesting that miR-275 regulates Vg-2 at the fed adult stage. The miR-275 tissue-specific results indicated high levels of miR-275 in unfed tick midgut, and expression was upregulated 2.72-fold in the ovary and 1.68-fold in the midgut after blood-feeding. The results suggested that miR-275 is predominantly involved in ovary development and blood digestion, further demonstrating that miR-275 has an important role in egg development. In addition, approximately 6 days after blood-feeding, most H. longicornis females began to lay eggs and gradually lost weight. However, the weight of female ticks with Ant-275 treatment decreased very slowly, the egg laying period was prolonged approximately 2 days, and the final weight was heavier than that of the control groups. These abnormal results suggest that the blood was not digested entirely in the midgut; depletion of miR-275 slowed blood digestion. The blood meal provides a variety of nutrients for ovary development and egg laying; thus, arrested ovary development and lower egg number may be caused by digestive disorders. Therefore, blood digestion and ovary development are inseparable. However, blood intake of female ticks was not affected, suggesting that miR-275 carries out its functions after blood meal. miR-275 has been found in multiple arthropods , especially hematophagous species. Therefore, this widespread phenomenon of miR-275 may be essential for growth and development in arthropods.
In mosquitoes, several specific miRNA-regulated blood digestion and egg development processes have been identified [24, 26], but there is little research about miRNAs in ticks. Both miR-5316b and miR-5331 are novel miRNAs in eggs, suggesting that these two miRNAs are indispensable for egg development. miR-307, miR-5317 and miR-5318 were only detected in female ticks, indicating they may be involved in reproduction . This work is the first to elucidate the function of miRNAs in H. longicornis ticks. These results provide reliable information for further analyses of egg development and blood digestion in ticks. In this work, we showed that miR-275 targeted Vg-2 and regulated ovary development and blood digestion, indicating that miR-275 has similar functionality in H. longicornis and mosquito; thus, specific miRNAs are important to hematophagous arthropods and may have similar functions. Therefore, our study may provide a new way to control the reproduction of ticks.
To our knowledge, this is the first report showing that miR-275 targets Vg-2 and regulates ovary development and blood digestion in H. longicornis. Our data suggest that miR-275 and Vg-2 are essential for reproduction and blood digestion of female ticks. Thus, these results suggest new methods for researching the development and reproduction of ticks.
This study was financially supported by the NSFC (31572511); the National Key Research and Development Program of China (2016YFC1202000, 2016YFC1202002); the 973 Program (2015CB150300); ASTIP, FRIP (2014ZL010), CAAS; and NBCIS CARS-38.
Availability of data and materials
The datasets supporting the conclusion of this article are included within the article.
GYL designed this study and critically revised the manuscript. JWH participated in its design, coordination and revised the manuscript. JL, XCL, FW and ZW participated in sample collection. ZC, QYR, JXG, QYC, JXL, YH and HW performed the experiments, data analysis, and drafted the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
This study was approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.
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