Researches On The Funcational Factors Of Male Gonad From Two Ixodid Ticks

Mating is an important prerequisite for tick reproduction,and initiates a cascade of physiological changes in female that eventually leads to oviposition. Male accessory gland (MAG) plays an important role during mating,which protects and activates the spermatozoa,promotes ovary development and vitellogenesis, exerts inductivity effects on female reproductive physiological activity,induces or accelerates rapid feeding,ovulation,oviposition and many other functions. The morphological features and the secretion biochemical characteristics of MAG are introduced briefly,and the present research situation of the functional factors are systematically reviewed,focuses on the functional factors associated with the sperm capacitation,female engorgement,ovary development and vitellogenesis. Finally,the future research is prospected, so as to develop ideas for this tick research field.The present study focuses on two ixodid tick species, Haemaphysalis longicornis and Dermacentor silvarum, which are wide spread in Hebei Province. Injection of hemocoel, Enzyme-Linked Immunosorbnent Assay (ELISA) were performed to study the function of secretion of H. longicornis male sex gland. The techniques of construction of cDNA library, rapid amplification of cDNA end(RACE), reverse transcription-PCR (RT-PCR) and Real-Time PCR were performed to amplify the full-length sequence of engorgement factor (EF) and analysis life-stage and tissue expression of EF. Liquid Chromatography -Mass Spectrometry (LC-MS)were performed to analysis the proteomics of sex gland of fed males. The above results would be basic for digging the function factors which play a role to trigger the mating responses of females and further developing anti-tick vaccine. Aiming to further understand the characteristics of EF and support the study of sequence conservativeness of it, the techniques of construction of cDNA library and RACE were also conducted to isolate the full-length sequence of EF of D. silvarum. The major results for the present study are as followed:1. H. longicornis(1). The extracts of sex gland from engorged male could stimulafe virgin females feeding, from slow feeding phase turn to rapid feeding phase, be engorgement quickly and then detaches , shown on engorgement period shorter, rate of engorgement higher, the influence on engorgement weigh was significant.(2). The extracts of sex gland from engorged male could stimulafe the development of ovarian, advanced the fat body synthesis vitellogenin, and forward the ovarian uptaked vitellin.(3). The virgin females injected with different doses of sex gland from engorged male has not significant differences in the engorged weight, ovarian weight, fat body synthesis vitellogenin and ovarian uptake vitellin. Only the highest dose experiment have differences in engorged weight and synthesis vitellogenin.(4). According to the results of injection of male ticks gland extract into hemocoe similar with the mating, in the mating the function substances for mating from male ticks were transported into the female's reproductive tract. In this experiment the extracts were transported into the hemolymph of female ticks. Therefore infer that the pathway of male mating function substances in the female tick was when transport into the reproductive tract with sperms then passes through the reproductive tract, into the hemolymph, circulation through the hemolymph to reach the target tissue and functioning.(5). The high quality cDNA library of sex gland from engorged male was successfully constructed, The titer of primary library was 1.71×106, and the amplified library's was 2.8×108.(6). The full length sequence of engorgement factor HlEFαwas successfully amplified (HQ908090), The HlEFαlength is 647 bp, open reading frames starts at 116 position and ends at 572 position, and encods 152 amino acids.(7). Eggs developmented for ten days, larval, nymph, female and male in the unfed period were not produced HlEFα. The tissues from virgin female fed 96 h were not detected HlEFα, and there are little changes on the HlEFαcontents of ovarians before and after mating.(8). Feeding or gonads developed maturation is a necessary condition for HlEFαsynthesis, it is inferred that male ticks testis / vas deferens were synthetic part.(9). A number of gene sequences were obtained from the cDNA library of sex gland from engorged male by selected randomly.(10). Part of this tissue's proteins during engorged period were detected by LC-MS analysis and comparison of the total protein from gonads of engorged male. Proposed future experiments could use two-dimensional electrophoresis for separation preliminary before the LC-MS analysis.2. D. silvarum(1). The high quality cDNA library of sex gland from engorged male was successfully constructed, calculate the titer of primary library was 2.17×106, and the amplified library's was 3.5×108。(2). The sequence of engorgement factor DsEFαwas successfully amplified (HQ908089), The DsEFαlength of 672 bp, open reading frames starts at 127 position and ends at 580 position, and encods 151 amino acids.(3). A number of gene sequences were obtained from the cDNA library of sex gland from engorged male by selected randomly.(4). Aligement of the deduced amino acid sequences of EF among H. longicornis, D. silvarum, R. appendiculatus, A. hebraeum, D. variabilis suggest that the similarity of the five sequences coincide with the evolutional classification, i.e. higher similarity were found in closer evolutional relationship. There are higher conservative between Dermacentor and Rhipicephalus, this is related they belong to the same evolutionary branch.