Analysis On The Hemolymph Proteins And Lipids Of Haemaphysalis Longicornis (Acari: Ixodidae)

Ticks are special kinds of arthropod.They transmit a series of infectious pathogens and cause serious damage to human,livestock and wildlife.Tick hemolymph is the site of metabolic,knowledge of tick hemolymph is very important for deeply research on tick physiology,biochemistry,pathology,toxicology,as well as cell culture.Experiments were conducted on the hemolymph of female Haemaphysalis longicornis Neumann,which was distributed widely in China,including protein and lipid and their change during different developmental stages.Using many modern biological technologies and the methods of the cross correlative realm,such as Bradford,SDS-PAGE,Western-blot and GC/MS etc.The present results will provide foundation for further investigations on tick physiological mechanism.The main results were as follows:1.Base on hemolymph of low quantity,condensation,dark,degeneration,pollution, two different collection methods were established along with development.For unfed female tick,the integument edge was cut and the hemolymph were collected;for feeding females, hemolymph was collected by cutting legs at the coxal-trochanteral joint.2.The protein level of the hemolymph varied obviously during the female developmental stages.The concentration of protein was low(5.918mg/mL),and increased rapidly at the early stage after attachment.At the mating stage,the protein level continuously increased,and reached its maximum level on the day of engorgement(17.834 mg/mL). Compared with 4th day after engorgement,the hemolymph protein contents declined signficantly,when the oviposition began,while there was not great difference when compared with 2nd day after engorgement3.The results of SDS-PAGE showed that the hemolymoph protein components were very complicated.At the unfed stage,5 main protein bands were detected in female hemolymph,which molecular weight were 246KD,103 KD,65KD,54KD and 48KD respectively;From the onset of feeding to mating stages,protein bands increased obviously, there were 5 new bands appeared,which molecular weight were 348KD,95KD,80KD, 62KD and 58KD.At mating stage,one new band appeared and its molecular weight was 69KD,and increased again at the engorgement stage,the protein added 10 bands than mating stage,which were 320KD,222KD,192KD,165KD,134KD,112KD,78KD,71KD,60KD and 35 KD;the hemolymph possessed the maximum protein components at the stage from engorgement to the beginning of oviposition,and invariable at these stages.4.Hemolymph vitellogenin(Vg) was identified by Western-blot,detected by 5%～15% graded electrophoresis,stained by Coomassie Brilliant Blue R-250,and analysized by image acquisition and analysis system,the molecular weight of Vg was 487KD.5.Hemolymph lipids were detected by GC/MS.The results showed there are five fatty acids,which were 14-methyl-pentadecane acid,palmitic acid,3-styrene-acrylic acid, 16-methyl-heptadecanoic acid and stearic acid respectively.These fatty acids were homology at different developmental stage,but their percents were different.