Ovary Development Of Reticulitermes Spp.and Related Gene Expression Of Parthenogenetic Eggs

Reticulitermes aculabialis and R.flaviceps are the major harmful termite species in China.In this study,the experiment was divided into four groups:R.aculabialis female-famale pairs(RaFF),R.aculabialis female-male pairs(RaFM),R.flaviceps female-famale pairs(RfFF)and R.flaviceps female-male pairs(RfFM).Through the method of microexamination,fluorescence staining,paraffin section,RT-PCR and statistical analysis,we compared systematacially the number of laid eggs and micropyles,ovaries development of primary reproductive,embryogenesis of unfertilized eggs and expression level of 8 genes which related to development between two species.In order to reveal the influence factors and regulating mechanism of parthenogernesis,we compared R.aculabialis which have parthenogernesis while R.flaviceps which have no parthenogernesis in both morphological and genetic level.The conclusions are as follows:(1)According to different mature level of the largest oocyte,we divided the ovaries into five stages:previtellogenic oocytes,early vitellogenic oocytes,late vitellogenic oocytes,mature oocytes and oocytes in the oviduct(stages A-E,respectively).The total percents of RfFF and RfFM which in A and B stages respectively were 53.33%and 58.33%,while in RaFF and RaFM the total proportion of ovaries in C and D stages respectively were 99.98%and 75%.Therefore we found that the ovaries of R.aculabialis have better development than R.flaviceps.In RaFF and RfFF,the femal alates firstly laid eggs 35.12±2.59 and 26.6413.78 days after colony foudation,and the monthly average number of one colony collected eggs respectively were 18.24±3.18 and 11.53±4.51 in August,11.16±4.26 and 3.67±1.24 in September.The result suggested that R.aculabialis female-female laid more eggs than R.faviceps during two months and.flaviceps laid more eggs in August rather than September(P<0.05).The number of micropyles in RaFF,RaFM,RfFF and RfFM respectively were 6.31±1.89,8.18±3.22,8.15±2.67 and 8.43±3.05.The number of micropyles in RaFF were significantly more than other groups(P<0.05),but there was no significant difference among the other groups.(2)There was only one nucleus in mature oocyte of ovaries in R.flaviceps and R.aculabialis,while just laid eggs have 2-5 nuclei,indicating that activation and 1-2 times cleavage of oocyte occured during ovulation.The number of eggs nucleus of R.aculabialis unfertilized eggs were 16-22(5 h),35-42(10 h),51-60(24 h)and 67-84(48 h).The 3-5 d embryo formed embryonic disc,7-9 d eggs formed embryonic band and the embryonic band elongated 10-15 d after laid.The umber of nuclei of R.flaviceps unfertilized eggs were 6-10(5 h),28-37(48 h),71-85(5-7 d),respectively.The cleavage rate of R.flaviceps was significantly lower than R.aculabialis.86%of R.flaviceps unfertilized eggs stoped development during formation of the blastoderm and the yolk cell massive gathered in the centre of egg.(3)Compared with female alates and ovaries in same species of RfFF and RaFF,exprssion of csf in R.aculabialis ovaries and the other seven genes(cdc2,cyclin B,cyclin B3,mapkapk,cdc42,tnk2 and ssp4)in two species ovaries were significantly higher than alates(P<0.05),implying that these genes were up-regulated in ovaries.In addition,seven genes(cdc2,csf,cyclin B,cyclin B3,mapkapk,cdc42 and ssp4)in two species ovaries and tnk2 in R.aculabialis ovaries were significantly higher than laid-eggs of the same species,indicating that these maternal genes maybe degraded in laid-eggs.Compared with two different species,we found that cyclin B and cyclin B3 expression in R.aculabialis female were significantly higher than R.flaviceps female.The expression of six genes(cdc2,cyclin B,cyclin B3,mapkapk,cdc42 and thk2)in R.aculabialis ovaries were significantly higher than R.flaviceps ovaries,on the contrary csf gene in R.aculabialis ovaries were significantly lower than R.flaviceps ovaries,so we suspected that these genes played a important role in ovary development and oocyte maturation.we also found that tnk2 expression in R.aculabialis eggs were significantly higher than R.flaviceps eggs,suggesting that tnk2 gene may be relative to embryo development of termite unfertilized eggs.