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The salivary glands are vital to the biological success of ticks and they are a major route of pathogen transmission. Tick salivary glands undergo remarkable growth and differentiation during the blood-feeding period. MicroRNAs (miRNAs) are noncoding small RNA molecules found in diverse organisms that regulate gene expression at the post-transcriptional level. To explore transcriptional differences in the miRNAs of fed and unfed tick (Haemaphysalis longicornis) salivary glands, we investigated small RNA (sRNA) transcriptomes derived from the salivary glands and made a comparative analysis of miRNA profiles related to tick blood-feeding in the salivary glands. We generated two small RNA libraries from the salivary glands of unfed and fed H. longicornis, and obtained 14.8 and 10.3 million reads of 18-30 nt, respectively. The unfed-specific sRNAs were clearly richer than the fed-specific sRNAs in terms of the unique and total sRNAs. Overall, 769 conserved miRNA families were found in unfed samples, whereas 440 conserved miRNA families were found in fed samples. Six of the ten most abundant miRNA were found in both the unfed and fed tick salivary glands, i.e., miR-1, miR-375, bantam, miR-184, miR-739, and miR-263a. We found that known miRNA homologs displayed a wide variety of expression profiles in unfed and fed tick salivary glands. After blood-feeding, 162 known miRNAs were upregulated. The six main upregulated miRNAs were mir-1810, mir-2138, mir-2140, mir-425*, mir-429, and mir-516*. Likewise, 231 known miRNAs were downregulated after blood-feeding. The six main downregulated miRNAs were miR-2941-1*, miR-10-5p, miR-2973, miR-1183, miR-4006b-5p, and miR-881. We found that distinct microRNA profiles in the salivary glands of H. longicornis were relating to tick blood feeding. The differential expression of miRNAs in unfed and fed tick salivary glands supported their involvement at new levels in the regulation of tick blood-feeding. Our data provide an important resource for a more detailed functional analysis of miRNAs in this species.

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The present study demonstrates that the subcutaneous administration of Neospora caninum dense granule protein 7 (NcGRA7) entrapped in liposomes coated with mannotriose strongly induces the parasite-specific T-helper type 1 immune response and humoral antibody in mice. Although anti-NcGRA7 immunoglobulin G1 antibody production was induced in mice injected with NcGRA7 alone, the dams and offspring were never protected from N. caninum infection. The immunization of mice with liposome-entrapped NcGRA7 before pregnancy resulted in increased offspring survival and decreased the infection rates in the brains of dams after parasite infection at 6 to 9 days of gestation. In conclusion, oligomannose-coated liposome-entrapped NcGRA7 can be used as a new type of effective vaccine to control neosporosis.

Background: Apoptosis is fundamental in maintaining cell balance in multicellular organisms, and caspases play a crucial role in apoptosis pathways. It is reported that apoptosis plays an important role in tick salivary gland degeneration. Several different caspases have been found in ticks, but the interactions between them are currently unknown. Here, we report three new caspases, isolated from the salivary glands of the tick Rhipicephalus haemaphysaloides. Methods: The full-length cDNA of the RhCaspases 7, 8 and 9 genes were obtained by transcriptome, and RhCaspases 7, 8 and 9 were expressed in E. coli; after protein purification and immunization in mice, specific polyclonal antibodies (PcAb) were created in response to the recombinant protein. Reverse-transcription quantitative PCR (RT-qPCR) and western blot were used to detect the existence of RhCaspases 7, 8 and 9 in ticks. TUNEL assays were used to determine the apoptosis level in salivary glands at different feeding times after gene silencing. The interaction between RhCaspases 7, 8 and 9 were identified by co-transfection assays. Results: The transcription of apoptosis-related genes in R. haemaphysaloides salivary glands increased significantly after tick engorgement. Three caspase-like molecules containing conserved caspase domains were identified and named RhCaspases 7, 8 and 9. RhCaspase8 and RhCaspase9 contain a long pro-domain at their N-terminals. An RT-qPCR assay demonstrated that the transcription of these three caspase genes increased significantly during the engorged periods of the tick developmental stages (engorged larval, nymph, and adult female ticks). Transcriptional levels of RhCaspases 7, 8 and 9 in salivary glands increased more significantly than other tissues post-engorgement. RhCaspase9-RNAi treatment significantly inhibited tick feeding. In contrast, knockdown of RhCaspase7 and RhCas-pase8 had no influence on tick feeding. Compared to the control group, apoptosis levels were significantly reduced after interfering with RhCaspase 7, 8 and 9 expressions. Co-transfection assays showed RhCaspase7 was cleaved by RhCaspases 8 and 9, demonstrating that RhCaspases 8 and 9 are initiator caspases and RhCaspase7 is an executioner caspase. Conclusions: To the best of our knowledge, this is the first study to identify initiator and executioner caspases in ticks, confirm the interaction among them, and associate caspase activation with tick salivary gland degeneration.