Vitellogenesis And Hormonal Regulation Of Haemaphysalis Longicornis (Acari: Ixodidae)

Ticks represent a group of highly specialized bloodsucking arthropods that are obligate temporary parasites of mammals, birds, or reptiles. They are well known vectors not only transmitting a great variety of infectious pathogens, but also having a super reproductive ability, and causing serious damage to human and domestic animals. At present, the reproductive physiology of ticks has become one of the international focus research realms. Vitellogenesis and hormonal regulation are the essential contents of this realm. Based on the purification of the vitellin, a series of researches were conducted on the vitellogenesis and hormonal regulation of Haemaphysalis longicornis Neumann, which was distributed widely in China, including the physical and chemical properties of the vitellin and the vitellogenin, the protein sequence analysis, the localization of the vitellogenin synthesis, the dynamic changes of the vitellogenin synthesis and release and the effects of juvenile hormone and ecdysteriods. Many modern biological technologies and the methods of the cross correlative realm, such as tissue incubation, biological chemistry, analytical chemistry, immunocyte chemistry, ELISA, GC/MS and mass peptide mapping etc., were applied in these researches. The present research could be of important scientific significance in elucidating the vitellogenesis and regulation mechanism and revealing the reproductive biological laws, the adaptive strategy and the physiological regulative mechanism in ticks.The vitellin (Vn) was firstly purified from eggs of H. longicornis, which was only one kind of protein. The Black B and periodic acid-Shchiff (PAS) could stain Vn, and it revealed that Vn was a hemoglycolipoprotein. The isoelectric point of Vn was 5.4 and its relative molecular weight was 370 KD. Vn was composed of eight polypeptides with the relative molecular weight of 112, 103, 80, 78, 71, 68, 62 and 52 KD respectively. N-terminal sequence analysis of the third Vn subunit revealed 15 amino acids in the following order: YSVLPVYLPCFYEKC. A sequence was unobtainable from the first Vn subunit. The MALDI-TOF-MS technology was firstly applied on the research of Vn protein sequence. The peptide mass fingerprinting (PMF) of the eight Vn subunits was obtained. The software (MASCOT) was used to compare the PMF of the eight Vn subunits against a protein databasemaintained by the National Center for Biotechnology Information (NCBI) with online. A precise match was not found in this database. The result revealed that the Vn subunits were the new proteins, which had not been recorded in the data library of NCBI.Carbohydrates and Lipids were detected by GC/MS in Vn. Carbohydrates were predominantly mannose and glucose with a small amount of rhamnose, ribose, arabinose, xylose, galactose, sorbose, N-acetylglucosamine and N-acetylgalactosamine. Lipids were mainly 13-octadecenoic acid and octadecanoic acid with some pentadecanoic acid and 8, 11 -octadecadienoic acid.In H. longicornis, the fat body consisted of one basic cell type, the trophocyte. In addition, a distinctly different cell type, the nephrocyte, was often found attached to the trophocyte. The ultrastructure of the fat body trophocytes at vitellogenic stage appeared consistent with intense protein synthesis. The results of PAGE and SDS-PAGE showed that the fat body protein components were very complicated. At the unfed stage, the fat body protein components were few, increased at the early stage of attachment, invariable at the mating stage, and increased again at the engorgement stage, and then at the early stage of oviposition, the partial protein components became disappeared. The fat body possessed the maximum protein components at the stage from engorgement to the beginning of oviposition. The fat body was the site of Vg synthesis in H. longicornis, confirmed by Western-Blot and Immunoelectron Microscopy, and not only in the central fat body but also in the peripheral fat body, the trophocytes synthesized Vg.When the female began to feed, the hemolymph protein components increased gradually, became the best rich at the engorgement stage, and decreased at the early stage of oviposition. All of the Vn subunits could be found in the hemolymph except the sixth subuinit. The subunits, PI, P4, P5, began to appear at the engorgement stage;and the subunits, P2, P3, P7, P8, had been present at the early stage of attachment. The Vg was identified by Western-Blot with antibody against Vn in hemolymph of the female, and the relative molecular weight of the Vg was 480 KD. The Vg content of the hemolymph was low at the early stage of attachment, increased distinctly at the mating stage and increased continuously at the engorgement stage, and then reached a high level on the 4th day after engorgement. The Vg content decreased from the beginning of oviposition to the 2nd day after oviposition, butincreased again and reached the maximum level rapidly on the 3rd day after oviposition, subsequently, the Vg content decreased gradually and declined to a low level on the 10th day after oviposition.The protein level of the ovary varied obviously during the female developmental stages. The concentration of protein was low, and increased rapidly at the early stage after attachment, which was about 9 times than that of the unfed female. At the mating stage, the protein level continuously increased, subsequently, it rose 3-5 times compared with the previous stage, and reached its maximum level on the day of oviposition. After oviposition, the protein level began to decline. In unfed females, only one main ovary protein band was detected. At the early feeding and mating stages, the protein band multiplied obviously, and maximized on the day of engorgement. Fifteen main protein bands of the three stages were the same. Subsequently, the main protein bands changed distinctly. Five protein bands were disappeared, and the relative molecular weights of them were 97 KD, 91 KD, 64 KD, 54 KD and 53 KD respectively. The predominant protein bands became the eight subunits of Vn.Ecdysteriods but not juvenile hormone caused an obvious increase in hemolymph Vg concentration, and had effects on synthesis and release of Vg in H. longicornis. Injection of 20-E, the doses of 2, 5, 10 ug /g body weight enhanced the hemolymph Vg concentration distinctly for the females at the mating stage, and the doses of 5, 10 |ig /g body weight had the same effect on that of the engorgement females. Topical application of JH III had no significant effect on the hemolymph Vg concentration, the doses of 20, 50, 100 jig /g body weight produced only a slight increase in Vg titers that was not statistically different from the acetone control for the females at the engorgement stage.Juvenile hormone and ecdysteriods could affect egg development of H. longicornis profitably. Topical application farnesol (a juvenile hormone analogue, JHA), the doses 50 \ig enhanced the protein concentration and the Vn level of the ovary on the day of engorgement obviously. The effect of the doses over lOOug was toxic. Farnesol had no significant effect on the protein components of the ovary. The ovaries of the females on the day of engorgement were cultured in vitro by TCI99, which contain 20-E with a gradient of concentrations. The 1.00 ng/uL dose could enhance the protein concentration and promote the ovary to absorb vitellogenin. However, the 5.00 ng/uL dose played a passive roles. The 2.50 ng/uL and 5.00ng/uL doses changed the migration rate of Vn in PAGE. The above results suggested that juvenile hormone and ecdysteriods control reproduction together and both of them affect vitellogenesis in H. longicornis.