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Abstract Background Haemaphysalis longicornis is an obligate hematophagous ectoparasite that transmits a variety of pathogens causing life-threatening diseases in humans and animals. Paramyosin (Pmy) is not only an invertebrate-specific myofibrillar protein but also an important immunomodulatory protein. Therefore, it is one of the ideal candidate antigens for vaccines. Methods We conducted two vaccine trials to evaluate the protective efficacy of Pmy recombinant protein (rPmy) and peptide vaccine (KLH-LEE). Each rabbit was immunized with three doses of rPmy or KLH-LEE adjuvanted with Freund’s complete/incomplete at 500 μg/dose at 2-week intervals before challenge with 40 female H. longicornis/rabbit. PBS plus adjuvant, Trx or KLH was used as control group. The antibodies of rabbits were detected by ELISA. Then, female ticks were fed on the rabbits until detachment. Results ELISA results showed that both vaccines induced rabbits to produce antibodies. Compared with the Trx group, the engorgement weight, oviposition and hatchability of the rPmy group decreased by 8.87%, 26.83% and 38.86%, respectively. On the other hand, engorgement weight, oviposition and hatchability of female ticks in the KLH-LEE group correspondingly resulted in 27.03%, 53.15% and 38.40% reduction compared with that of the KLH group. Considering the cumulative effect of vaccination on the evaluated parameters, results showed 60.37% efficacy of the rPmy vaccine formulation and 70.86% efficacy in the KLH-LEE group. Conclusions Pmy and particularly epitope LEE have potential for further development of an effective candidate vaccine to protect the host against tick infection. Graphic abstarct

Abstract Background The tick Haemaphysalis longicornis exhibits two separate reproductive populations: bisexual and parthenogenetic, which have diploid and triploid karyotypes, respectively. The parthenogenetic population can undergo engorgement without copulation and produce viable female-only offspring with a longer incubation period than the bisexual population. Three enzymes, cathepsin B, cathepsin D and acid phosphatase, were found to be involved in vitellin degradation during the embryonic development of bisexual H. longicornis. However, the expression and activity profiles of these enzymes during the embryonic development of parthenogenetic ticks remain unknown. In the present study, the transcriptional expression profile, enzyme activity and roles in embryogenesis of the three enzymes during the embryonic development of parthenogenetic H. longicornis were investigated. Methods Quantitative real-time polymerase chain reaction (qPCR) and fluorescence detection were used to analyze the dynamic changes in the three enzymes during embryogenesis. The roles of the three enzymes during embryogenesis were also explored using RNA interference (RNAi). Results The three enzymes were all expressed during embryonic development in parthenogenetic H. longicornis. The expression of cathepsin B was highest on day 15, whereas that of cathepsin D was highest on day 3 and the peak of acid phosphatase expression occurred on day 9. The activity of cathepsin B was highest on day 3 and lowest on day 5, then gradually increased and remained stable. Cathepsin D activity was highest on day 1 and showed a gradually decreasing trend, whereas acid phosphatase showed the opposite trend and reached a peak on day 23. RNA interference experiments in engorged female ticks revealed that there was no significant difference in the number of eggs laid, but the hatching rate of the eggs was significantly decreased. Conclusion The three enzymes all play important roles in embryonic development of H. longicornis, but the expression patterns and changes in the activity of the enzymes in the bisexual and parthenogenetic populations are different. The results will help a better understanding of the similarities and differences underlying embryonic development in the bisexual and parthenogenetic populations and contribute to the future exploration of the development of the parthenogenetic population of H. longicornis.

Abstract Background The important roles of microbial flora in tick biology and ecology have received much attention. Dermacentor marginatus and Dermacentor reticulatus are known vectors of various pathogens across Europe, including Slovakia. However, their bacterial microbiomes are poorly explored. Methods In this study, bacterial microbiomes of field-collected D. marginatus and D. reticulatus from Slovakia were characterized using 16S rRNA high-throughput sequencing. Results Different analyses demonstrated that the D. marginatus and D. reticulatus microbiomes differ in their diversity and taxonomic structures. Furthermore, species- and sex-specific bacteria were detected in the two species. A possible bacterial pathogen “Candidatus Rhabdochlamydia sp.” was detected from D. marginatus males. Among the observed bacteria, Rickettsia showed high abundance in the two species. Several maternally inherited bacteria such as Coxiella, Arsenophonus, Spiroplasma, Francisella and Rickettsiella, were abundant, and their relative abundance varied depending on tick species and sex, suggesting their biological roles in the two species. Conclusions The bacterial microbiomes of field-collected D. marginatus and D. reticulatus were shaped by tick phylogeny and sex. Maternally inherited bacteria were abundant in the two species. These findings are valuable for understanding tick-bacteria interactions, biology and vector competence of ticks.

Abstract Background Three main enzymes including cathepsin B, cathepsin D and acid phosphatase are involved in vitellin degradation, which is a major biochemical event of the embryonic development and can provide nutrients and metabolites for tick embryos. In the present study, the mRNA expression profiles and enzymatic activity of cathepsin B, cathepsin D and acid phosphatase were investigated during embryonic development in the tick Haemaphysalis longicornis. Results The results revealed that all three enzymes were expressed throughout embryonic development. Both cathepsin B and acid phosphatase transcripts were accumulated during the first four days. Cathepsin B reached its highest expression on day 5, whereas the peak expression of acid phosphatase and cathepsin D occurred on day 11. The highest activity of cathepsin B was observed on the first day of egg development, whereas cathepsin D reached its highest activity on day 13. Acid phosphatase activity increased gradually during the first five days and then remained stable until the end of egg development. Conclusions Three enzymes were expressed and activated in eggs, and also presented different dynamic changes with the development of embryos. The profiles of both mRNA expression and enzymatic activity of these enzymes indicate that they are controlled orderly and play multiple roles during embryonic development in ticks.

Abstract Background Haemaphysalis longicornis is a blood-sucking ectoparasite that can cause diseases by transmitting some pathogens to humans and animals. Paramyosin (Pmy) is an immunomodulatory protein, which plays an important role in immune reactions against parasites. In this study, we evaluated the immune protection elicited by recombinant plasmids encoding H. longicornis Pmy in rabbits. Results Rabbits vaccinated with pcDNA3.1(+)-Pmy developed high level of IgG compared to control group, suggesting that humoral immune response was induced by vaccination. On the fourth day after fed on the rabbit, some female adults died and the mortality rate from pcDNA3.1(+)-Pmy group (27.31%) was significantly higher than that of the control group (P

Abstract Background The survival of overwintering ticks, is critical for their subsequent population dynamics in the spring, and consequent transmission of tick-borne diseases. Survival is largely influenced by the severity of the winter temperatures and their degree of cold hardiness at the overwintering stage. The bush tick Haemaphysalis longicornis, is widely distributed in China, and can transmit various pathogens that pose serious medical/veterinary problems. In the present study we investigated the effect of low temperature stress to tick survival, super-cooling point and body content of water, glycerol and total protein. Methods After various temperature acclimations, the super-cooling point was measured by Ni/CrNi-thermocouples with a precision temperature recorder. Water content was determined from weight loss of the sample exposed to 60°C for 48 h. Glycerol content was determined using Free Glycerol Reagent as directed by the manufacturer, and total protein was determined using the Bradford assay. Results The 50% mortality temperatures for the adults and nymphs were -13.7°C and -15.2°C, respectively; and the discriminating temperatures for the adults and nymphs were -16.0°C and -17.0°C, respectively. The super-cooling points of the adults and nymphs were -19.0°C and -22.7°C, respectively. The water content of adult H. longicornis decreased substantially after acclimation at 0°C for 10 d, whereas the nymphs decreased after acclimation at 0°C for 20 d, and the glycerol and proteins of both nymphs and adults were significantly increased (p