| Ticks are a group of ectoparasites with obligate feeding and strong reproduction ability,and can transmit a great variety of pathogens,which cause great damages to human and livestock.Haemaphysalis longicornis is distributed in China and many other countries,it can be divided Haemaphysalis longicornis bisexual population(HLBP)and Haemaphysalis longicornis parthenogenetic population(HLPP)according to different reproductive modes.HLPP is triploid,there are no male ticks in the whole life,and female ticks can complete the entire life cycle without fertilization.Embryonic development is an important physiological process during egg hatching into larva,vitellin(Vn)degradation is one of the important biochemical events,various kinds enzymes participate in Vn degradation and provide important nutrients.Temperature is an important ecological factor that affects enzyme activity,so it is of great significance to study the effect of temperature on enzyme and Vn degradation in the embryonic development of HLPP.This research took HLPP as object and applied modern biological techniques such as physiological,biochemical and immunology to study the effect of temperature on the development duration of HLPP,expression profiling and enzyme activity of cathepsin B(Cathepsin B,CB),cathepsin D(Cathepsin D,CD)and acid phosphatase(Acid phosphatase,AP)in different stages of HLPP embryo development at different temperatures,and dynamic changes of subunits during Vn degradation.The results aim at illustrating the rules of three enzymes and Vn degradation at different temperatures,and providing scientific basis for further elucidating the embryonic development mechanism of Haemaphysalis longicornis.1.The durations of hatching to larve at 16 ?,20?,26 ?,30 ? and 35 ? were observed and recorded respectively.The results showed that it took 24.6 d on average at 26?,110.6 d and 43.3 d respectively on average at 16? and 20?,21.6 d and 20.0 d are required on average at 30? and 35? respectively.2.Total RNA of eggs were extracted from 1 d,3 d,5 d,7 d,9 d,11 d,13 d,15 d and 17 d during the development of HLPP embryos at 16?,20?,26?,30? and 35? respectivelyfor CB and CD and AP analysis.The results showed that three enzymes were expressed at different temperatures,and temperature had effects on expression profiling of the three enzymes at different developmental stages,high temperature advances the days of maximum expression of CB and AP,and delays the appearance of days of maximum expression of CD.CB was expressed at the highest temperature on 15 d at 26?,and also expressed on 15 d at16? and 20?,the highest expression was at 30? and 35? both appeared on 7 d.The highest expression of CD at 26? and 20? appeared on the same day which was the 3 d,as the temperature increased,30 ? and 35 ? the highest expression levels of the enzyme appeared on 9 d and 15 d respectively.At 26?,AP had the highest expression on 9 d,at 16?and 20? it had the highest expression on 11 d,at 30? and 35?,AP had the highest expression on 5 d of embryo development.3.Total egg proteins from 1 d,3 d,5 d,7 d,9 d,11 d,13 d,15 d and 17 d during the development of HLPP embryos at 16 ?,20 ?,26 ?,30 ? and 35 ? were extracted respectively for CB and CD and AP enzyme activity analysis.The results showed that three enzymes were active at different temperatures.CB had the highest activity at 16?,20?,26?,30? and 35? on 17 d,9 d,3 d,17 d,and 15 d drespectively;CD reached the highest quantity on 1 d of embryo development at 26?,and the highest enzyme activity was at 13 d,17 d,13 d and 15 d at 16?,20?,30?,and 35? respectively;AP had the highest activity on 9 d at 26?,and the highest activity on 15 d,15 d,7 d,and 17 d at 16?,20?,30?,and35? respectively.4.The total egg proteins of 1 d,3 d,5 d,7 d,9 d,11 d,13 d,15 d and 17 d during HLPP embryo development at 16 ?,20 ?,26 ?,30 ? and 35 ? were extracted,and then performed by SDS-PAGE.The changes of intra-egg protein between 25 Kda and 170 Kda at different temperatures and different periods were analyzed of.The results showed that at 16?,20?,26?,30? and 35?,Vn was degraded to different degrees at 126 Kda;at 26 ? Vn at 70 Kda and 46 Kda also significantly changed with the embryo.Development gradually degraded,a new peptide appeared on the 13 d;at 16? Vn at 70 Kda also degraded,and a new peptide appeared on the 5 d;no new protein bands were found at other temperatures.Western-blot analysis was performed at 16?,20?,26?,30? and 35? at 1 d,5 dand 9 d eggs with Vn monoclonal antibodies,it was found that there are different degrees of degradation with embryo development at different temperatures;with the temperature increases,the faster the degradation,the lower the temperature and the slower the degradation.This study revealed the dynamic change of CB,CD and AP at HLPP different temperatures,clarified the effect of temperature on Vn degradation,provided an important scientific basis for in-depth elucidation of the embryonic development mechanism of Haemaphysalis longicornis. |
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