Studies On The Symbionts Of Haemaphysalis Doenitzi And The Pathogens Of Ixodes Scapularis

Ticks, as obligate blood-sucking parasitic arthropods, have a wide host range, so they achieve and spread many kinds of pathogens more easily and widely. The various pathogens can cause many terrible diseases and have serious damages on the health of humans and wildlife and livestock husbandry development. In addition to pathogens,there are numerous symbionts which are closely related to the life activity of host harbored in different tick species.Haemaphysalis doenitzi(Acari: Ixodidae), a three-host tick, distributed in many countries in Southeast Asia and Australia extensively. They harbored in the bird and small mammal mainly and carry various pathogens. Ixodes scapularis, as the primary vector of Borrelia burgdorferi, Anaplasma phagocytophilum and Babesia microti,spread all over the world especially in the middle and northeastern of United States. I.scapularis has been regarded as one of the most important tick species in public health in the United States.In this study, we first identified the symbionts species, analyzed their biological characteristics, assessed the population dynamics of symbionts in H. doenitzi and detected the effects of B. burgdorferi infection of I. scapularis on tick questing behavior by the methods of molecular biology, phylogenetics, morphology and behavior. Our study not only enrich the resource of tick symbionts and are helpful to more understanding of the biological characteristics and functions of Francisella-like symbionts, but also reflect the correlation between pathogens and tick behavior preliminarily. Hence, this study laid a good foundation for the research on the mutualism mechanism of ticks and their symbionts and the interaction between pathogens and ticks in the future. It provides the theoretical basis for developing the new methods for tick control. The main results of the research are as following:1. By constructing bacterial 16 S rRNA gene libraries of H. doenitzi collected from the field and PCR-RFLP analyses, we detected several microorganism in H.longicornis, including Sphingomonas, Acinetobacter and a novel Francisella-like symbiont(FLEs-Hd). The phylogenetic analysis showed the FLEs-Hd wasphylogenetically closely related to the Francisella-like symbionts of Hyalomma marginatum marginatum and Rhipicephalus sanguineus.2. The biological characteristics of FLEs-Hd were detected by diagnose PCR and transmission electron microscopy(TEM). Results indicated that FLEs-Hd had100% infection rate in the field colony of H. doenitzi and a perfect vertical transmission under laboratory conditions, and that it was distributed in ovaries,malpighian tubules, salivary glands and midguts of the ticks, suggesting that FLEs-Hd presumably is a crucial symbiont of the host without specific tissue tropism.3. The population dynamics of FLEs-Hd in the whole life cycle of H. doenitzi were estimated by real-time qPCR. The significant drop in the density of FLEs-Hd from egg to larval stage was probably due to the bottleneck effect during vertical transmission. The density of FLEs-Hd increased obviously after engorgement among larvae, nymphs and males stages, indicating that FLEs-Hd might be associated with host engorgement. In unfed females stage, the density of FLEs-Hd always kept in a lower level and increased significantly at the last day before feeding, demonstrating that FLEs-Hd might be closely related to the reproduction of females.4. In order to illustrate the impact of FLEs-Hd in the process of tick reproduction,the population dynamics of FLEs-Hd in different tissues of H. doenitzi were estimated using real-time qPCR. The densities of FLEs-Hd in ovary and malpighian tubules was higher than them in midgut and salivary glands. In ovaries, the density of the symbionts was higher at the initial feeding stage(2nd day of feeding) and at almost engorged female(4th day of feeding) and then dropped to a stable lower level at next stages. In fact, the steps of multiplication coincide with developmental phases of ovary. Simultaneously, the growth of symbionts in midgut was observed at the beginning of vitellogenin synthesis, suggesting that the FLEs-Hd might be related to the reproduction of the host. Therefore, it is speculated that the FLEs-Hd could be closely related to the initial development of ovary of the host and might provide some necessary nutrients or regulators to ensure the ovary development.5. The questing behavior and B. burgdorferi infection rate of I. scapularis from Texas and Maryland were assayed by the methods of behavior and molecular biology.The results exhibited that the I. scapularis from Maryland were more activity in questing behavior than them from Texas, and the prevalence of B. burgdorferi in I.scapularis from Maryland was also higher than them from Texas. Both of the results explained one of the reason about Lyme disease increasing gradually from south to north in America. But the prevalence of B. burgdorferi had less correlation to questing behavior of I. scapularis.