ABSTRACT
Successful completion of the molting process requires new epidermal growth and ecdysis of the old cuticle in Haemaphysalis longicornis (H. longicornis). MicroRNAs (miRNAs) participate in the development of organisms by inhibiting the expression of their target mRNAs. In this study, a novel tick-specific miRNA was identified and denoted hlo-miR-2 that serves as a novel regulator of molting events in H. longicornis nymphs by targeting a cuticular protein. The full length of this cuticular protein was first obtained and named it CPR1. A qRT-PCR analysis showed that hlo-miR-2 and CPR1 exhibit significant tissue and temporal specificity and that their transcription levels are negatively correlated during the molting process. CPR1, as a direct target of hlo-miR-2, was identified by a luciferase reporter assay in vitro. Agomir treatment indicated that the overexpression of hlo-miR-2 significantly reduced the protein expression level of CPR1, decreased the molting rate and delayed the molting time point in H. longicornis nymphs. RNA interference (RNAi) experiments demonstrated that CPR1 was significantly associated with the molting process in H. longicornis nymphs. Phenotypic rescue experiments convincingly showed that hlo-miR-2 participated in molting events by targeting CPR1 in H. longicornis nymphs. In summary, we present evidence demonstrating that miRNAs constitute a novel important regulator of molting events in addition to hormones. The described functional evidence implicating CPR1 in molting events contributes to an improved understanding of the distinct functions of the CPR family in ticks and will aid the development of a promising application of cuticular protein RNAi in tick control.
ABSTRACT
BACKGROUND: Insect-borne diseases could induce severe symptoms in human and clinical signs in animals, such as febrility, erythra, arthralgia and hemorrhagic fever, and cause significant economic losses and pose public health threat all over the world. The significant advantages of Luminex xMAP technology are high-throughput, high parallel and automation. This study aimed to establish a liquid bead array based on Luminex xMAP technology that was able to simultaneously detect multiple insect-borne pathogens. METHODS: Specific probes and primers to detect the nucleic acid of 10 insect-borne pathogens were designed. Probes were coupled with fluorescent carboxylated microspheres. The parameters of the system were optimized, including ratio of forward/reverse primers (1:2), hybridization temperature (50 °C) and duration (30 min) and quantity of PCR product (2 µl). The sensitivity and specificity of the system were also evaluated. Moreover mixed nucleic acid of 10 insect-borne pathogens, including Bluetongue virus, Epizootic hemorrhagic disease virus of deer, Coxiella burnetii, African swine fever virus, West Nile fever virus, Borrelia burgdorferi, vesicular stomatitis virus, Rift Valley fever virus, Ebola virus and Schmalenberg's disease virus, and 3000 clinical samples were tested for practicability. RESULTS: The optimized detection system showed high sensitivity, specificity and reproducibility. Each probe showed specific fluorescence signal intensity without any cross-hybridization for the other insect-borne pathogens tested, which included dengue virus, tick-borne encephalitis virus, Japanese encephalitis virus, Xinjiang hemorrhagic fever virus, spotted fever group rickettsiae, ehrlichiae and chikungunya virus. The limit of detection was 10 copies of target gene. Insect-borne pathogens were successfully detected among the 3000 clinical samples, and the results were consistent with those obtained using gold-standard assays or commercial nucleic acid detection kits. CONCLUSIONS: This optimized liquid array detection system was high-throughput and highly specific and sensitive in screening of the insect-borne pathogens. It was promising in detection of these pathogens for molecular epidemiological studies.
