The Role Of Circadian Clock Genes In Photoperiodic Response Of The Bird Cherry-oat Aphid,Rhopalosiphum Padi With Different Reproductive Modes

The bird cherry-oat aphid,Rhopalosiphum padi（L.）（Hemiptera:Aphididae）,is one of the most important wheat pests worldwide.This aphid not only damages by sucking plant directly and secreting honeydew causing sooty mould,but also by transmitting the barley yellow dwarf virus（BYDV）.Both types of damage significantly reduce the quality and yield of wheat crops globally.There are four different life cycles in R.padi.In a holocyclic life cycle,the cyclical parthenogenesis lineages would engage in sexual reproduction once per year under inductive conditions（short photoperiod and low temperature）,with generations of asexuality followed by the production of apterous viviparous females（named virginoparae）.The males and gynoparous females（gynoparae）would be parthenogenetically produced by virginoparae in secondary host（graminaceous plant）,then fly to primary host（Prunus）,and the oviparae would be parthenogenetically produced by gynoparae.The cold-resistant eggs produced by males and oviparae can withstand and survival the harsh winter weather conditions.However,the obligate parthenogenesis lineages can not produce sexual generation even in inducing conditions.Thus,the aphid provide an excellent model for studying the evolution of sex,empirically examining the relative costs and benefits of sexual versus asexual reproduction,and identifying the ecological cost of switching from asexual to sexual reproduction.There are differences in photoperiodic response between the cyclical and obligate parthenogenesis lineages,however the molecular mechanisms of the aphid photoperiodism has not yet been reported.In the current study,the roles of circadian clock genes in photoperiodic response of the bird cherry-oat aphid,Rhopalosiphum padi with different reproductive modes.The main results are as follows:1.Responses of R.padi with different reproductive modes to the low temperature and short photoperiodProlonged exposure to low temperature and a short photoperiod（LS）had negative effects on the offspring of anholocyclic aphids with regard to adult lifespan,total longevity,and fecundity compared with aphids maintained at a normal temperature and a long photoperiod（NL）.Holocyclic LS R.padi had longer developmental time at all larval stages,and had shorter adult lifespan,shorter total longevity,and lower fecundity than NL counterparts.The adult pre-reproduction period of gynoparae was significantly longer than that of virginoparae,whilst the total longevity of gynoparae was significantly shorter than that of virginoparae.Moreover,the net reproductive and gross reproduction rates,as well as the total fecundity,were around five-fold higher in virginoparae than in gynoparae.These results indicate that there is the short-term cost of sex.When maintained on the secondary host（T.aestivum）,gynoparae,males,and oviparae produced by holocyclic populations could survive,and gynoparae produced oviparae.However,under NL conditions,oviparae could not produce overwintering eggs on the secondary host,whereas a few eggs were generated by oviparae under LS conditions.2.Cloning and sequences analyses of 10 circadian clock genes from R.padiTwo Cry genes（Cry1 and Cry2）and two Tim genes（Timeout and Timeless）were successfully cloned from R.padi.The full lengths of another six circadian clock genes（Per,Clk,Cyc,Shy,Pdp1 and Vri）from R.padi were also obtained with RT-PCR and RACE techniques.Sequence analysis showed that these genes had the characteristics of respective gene family.Sequence similarity analysis showed that sequences of shaggy gene are highly conserved,and the deduced amino acid sequence of Tim1 and Per were greatly different with those of the corresponding protein sequences of other insect species of the Hemiptera（<35%）.Phylogenetic analysis indicated that the protein sequences of R.padi circadian clock genes were highly homologous with those of the corresponding protein sequences of the respective genes from Acyrthosiphon pisum,Myzus persicae and Diuraphis noxia.3.The influence of temperature and photoperiod on circadian expression of circadian clock genesAll the circadian clock genes（Cry1,Cry2,Clk,Per,Tim1,Tim2,Pdp1,Shy,and Vri）showed significant differences of their respective expression levels at circadian different time except the Cyc gene.Temperature had a critical effect on the circadian expression of some circadian clock genes.The transcript levels of Cry2,Clk,Cyc,Per and Vri genes at normal temperature were significantly higher than those at low temperature,whereas the transcript levels of Cry1 and Tim1 genes at low temperatures were significantly higher than those at normal temperature.There was no significant difference in the expression level of Shy,Tim2 and Pdp1 genes between normal temperature and low temperature.The expression of Cry1 gene under long photoperiod condition was significantly higher than that under short photoperiod conditions.Regardless of temperature,the transcript levels of the 10 circadian clock genes were significantly different at six different time under dark conditions.Temperature could significantly affect the expression of circadian clock genes.The transcript levels of Cry2,Clk,Cyc,Tim1,Tim2,Shy and Pdp1 genes at 24°C are significantly higher than those at 4°C.The expression levels of Clk,Cyc,Per,Tim2,Shy and Pdp1genes at 12°C were significantly higher than those at 4°C.Under the darkness condition,there were significantly differences in the transcript of some circadian clock genes between the head and the embryo.The circadian clock genes had different expression patterns under different temperatures.The transcript levels of Cry2,Per,Tim1,Tim2,Pdp1 and Shy genes in the head were significantly higher than those in embryos at 24°C.At 12°C,the expression levels of Clk,Per,Tim1,and Shy genes in the head were significantly higher than those in embryos.In general,there was no circadian clock gene in the embryo which had a significantly higher transcript level than in the head.4.Expression of circadian rhythm genes in photoperiodic response of R.padi with two different reproductive modesThe maternal environmental conditions would significantly affect the offspring’s response to short photoperiod and low temperature（autumnal inducing conditions）.A small amount of male could produce by the CP-LN population reared in long photoperiod and normal temperature which was induced for two generations.A large number of oviparae and male were produced under inducing conditions for three generations.However,under the same inducing conditions,CP-LL population reared in the long photoperiod and low temperature could produce a small amount of male in the first generation,and gynoparae,oviparae and male were produced in the second generation.These results indicated that when the mother was reared in low temperature conditions,oviparae and male would produce earlier under the induced conditions.The expression of per genes in the G₁ generation of the OP-LN population was not significantly different at different time.Under induction conditions,there were considerable differences in the expression of some circadian clock genes in four R.padi populations with different reproductive modes.Under long photoperiod and low temperature conditions,in addition to the Clk gene,the transcript levels of other nine genes were significantly different in the CP-LL and OP-LL populations,respectively.The expression levels of Cry1,Tim1 and Shy genes of CP-LL population were significantly lower than those in OP-LL population,while the transcript levels of Cry2,Cyc,Tim2,Vri and Pdp1 genes in CP-LL populations were significantly higher than those in OP-LL population.Under the inducing conditions,in addition to the Shy gene,there were significant differences in the expression levels of other nine genes between the CP-LL and OP-LL populations.The transcript levels of Cry1,Clk,Tim1,Tim2,and Pdp1 genes in CP-LL populations were significantly higher than those in OP-LL populations,while the expression levels of Per and Vri genes in CP-LL population were significantly lower than those in OP-LL population.5.The role of six core circadian clock genes in the aphid photoperiodismKnockdown of a circadian clock gene by RNA interference could significantly affect the expression of other circadian clock genes.Knockdown of Cry1 significantly reduced the expression levels of Cry2,Tim1,Clk,Cyc,Shy and Vri genes.Knockdown of Cry2 by reduced the expression levels of Tim1,Clk and Per genes and significantly promoted the transcription of Cry1.Knockdown of Clk significantly reduced the expression levels of Cry2,Tim1 and Cyc genes and promoted the significantly transcription of Per.Knockdown of Cyc significantly reduced the expression levels of Clk and Vri genes and promoted the transcription of Cry1,Pdp1 and Per.Knockdown of Per significantly reduced the expression levels of Cry2gene,and promoted the transcription of Cyc,and block the transcript of Tim1.Knockdown of Tim1significantly reduced the expression levels of Shy and Per gene,and promoted the transcription of Clk and Cyc.Nymphs of OP-LN population treated with dsRNA of circadian clock genes（Cry1,Cry2,Tim1,Per,Clk and Cyc）showed that no sexual generation was produced in offspring.However,Nymphs of CP-LN population treated with dsRNA of circadian clock genes（Cry1,Cry2,Tim1,Per,Clk and Cyc）showed that these genes had an important roles in the production of alate and apterous parthenogenetc female,male,gynoparae,and oviparae in offspring.Compared with the control group（injection dsGFP）,the number of apterous parthenogenetc female in treatment group（injection dsCry1,dsClk and dsCyc）was significantly decreased,and the number of male and oviparae was significantly increased.However,the number of apterous parthenogenetc female in treatment group（injection dsCry2,dsTim1 and dsPer）was significantly increased,and the number of male and oviparae was significantly decreased.Photoperiod could significantly affect growth and development of R.padi.The developmental time of the second,third and fourth instars larvae and the age of reproduction under the short photoperiod conditions were significantly longer than those in the long photoperiod condition under normal temperature condition.Temperature could also significantly affect the growth and development in R.padi.The developmental time for the first,second,third and fourth instars of larvae and the age of reproduction in low temperature were significantly longer than those in the normal temperature.Regardless of the rearing condition（long photoperiod and low temperature or long photoperiod and normal temperature）,when the same holocyclic lineges before induction were used before induction,the photoperiodic cycles required for the male production for the first time,oviparae production for the first time,and the generation of only the sexual generations in long photoperiod and low temperature conditions were considerable less than those in long photoperiod and normal temperature conditions.The results showed that the feeding conditions after induction had an important influence on the production of male and oviparae.In addition,when the aphids were reared in low temperature before induction,the low temperature condition promote earlier generation of the male and oviparae.In the four groups,CP-LN population which was transferred to long photoperiod and low temperature conditions after induction could produce more male and oviparae.CP-LL population which was transferred to long photoperiod and low temperature conditions after induction could produce more oviparae.After 11 and 10 days of induction under low temperature and short photoperiod conditions,no males were produced in both control groups injected with ddH₂O or dsGFP in the CP-LN population,but there were very few male in the three treatment groups which were injected with dsCry1 or dsClk or dsCyc.After15 and 14 days of induction,neither of the two control groups injected with ddH₂O or dsGFP produced oviparae in CP-LN population,whereas there were very few oviparae in the three treatment groups injected with dsCry1 or dsClk or dsCyc.After 24 and 23 days of induction for CP-LN population,both alate and apterous parthenogenetc female were found in the control groups,but no alate and apterous parthenogenetc female were found in the three treatment groups injected with dsCry1 or dsClk or dsCyc.After 12 days induction for the CP-LN population,there were few male in the two control groups,however,no males were produced in the three treatment groups which were injected with dsCry2 or dsTim or dsPer.After 16days for CP-LN population induction,there were few oviparae in the two control groups,whereas no oviparae was found in three treatment groups.After 25 and 26 days for CP-LN population induction,no alate and apterous parthenogenetc female was found in the control groups,but alate and apterous parthenogenetc female were produced in the three treatment groups.Different circadian clock genes had different effects on the photoperiodic timer process.After induction thirteen days for the CP-LN population,male were found in the control group and dsTim-injected treatment group.however none of males was produced in the dsCry2 and dsPer-injected treatment groups.After induction 17 days for CP-LN population,oviparae were present in two control groups,and no oviparae was produced in the dsPer-treated group,but very few oviparae were produced in the dsCry2 and dsTim-injected groups.6.The role of melatonin in the aphid photoperiodismIn this study,three arylalkylamine N-acetyltransferase genes（AANAT1,AANAT2 and AANAT3）and tryptophan 5-hydroxylase（TPH）gene were successfully cloned from R.padi.Q-PCR analysis revealed that levels of these genes expression were significantly affected by Zeitgeber time,and the mRNA expression of most of these genes had a robust diurnal oscillation with high at night phase and low at light phase.The results clearly revealed that the photoperiod could cause highly significant differences in the expression of the aphid genes AANAT1,AANAT3 and TPH.Temperature could affect daily oscillating expression and the amplitudes of expression curves of the four genes.The expression of AANAT1,AANAT2 and AANAT3 genes in constant dark conditions were significantly higher than that in constant light conditions.Light stimulus in constant dark conditions could significantly reduce the expression of AANAT1,AANAT2 and TPH genes.The expression of AANAT1,AANAT2 and TPH genes significantly increased after dark stimulus in constant light conditions.The findings also demonstrated that the mRNA expression patterns of these genes were daily oscillating and similar in head and embryo under constant dark conditions.Regardless of the feeding temperature at normal temperature or low temperature,the expression level of AANAT3 gene in the head was significantly higher than that in the embryo.There is an interaction between the circadian clock gene and melatonin,and the expression levels of the four melatonin synthesis-related genes were significantly increased of R.padi which were injected by dsCry1.Knockdown of Clk and Cyc genes significantly reduced the expression of AANAT1 and AANAT3genes.Knockdown of Tim and Per genes reduced the expression of AANAT3 gene significantly.Injection of exogenous melatonin could significantly reduce the expression levels of Cry1,Clk and Cyc genes,and also affect the expression of Cry2,Per and Tim1.Under inducing conditions,there were significant differences in the expression of four melatonin synthesis-related genes in different inducing generations of CP-LN and OP-LN populations.The expression level of the AANAT2 gene in the G₂-CP-LN population was significantly lower than that of the G2-OP-LN population.The expression levels of the AANAT3 gene in the R.padi populations（G₁-CP-LN,G₃-CP-LN,CP-LL and G₁-CP-LL）with cyclic parthenogenesis was significantly higher than that in the corresponding R.padi with obligate parthenogenesis.Under inducing conditions,the males and gynoparaes presented higher expression levels of AANAT and TPH genes compared with winged and wingless parthenogenetic females,and expression levels of these genes were lowest in oviparae.The injection of melatonin or knockdown of the AANAT3 gene could not cause the production of sexual generation in the cyclic parthenogenesis R.padi population which was reared in long photoperiod and normal temperature.And the treatments could not also cause the production of sexual generation in the obligate parthenogenesis R.padi population.Melatonin can significantly reduce the fecundity of R.padi.Nymphs of CP-LN population treated with melatonin and dsAANAT3 showed that melatonin and AANAT3 had an important roles in the production of alate and apterous parthenogenetc female,male,gynoparae,and oviparae in offspring.Compared with the control group（injection ddH₂O）,the percentage of apterous parthenogenetc female in treatment group（injection meletonin）was significantly decreased,and the percentage of male was significantly increased.However,the percentage of apterous and alate parthenogenetc females in treatment group（injection dsAANAT3）was significantly increased,and the percentage of gynoparae and oviparae was significantly decreased.Nymphs of CP-LN population treated with dsRNA of circadian clock genes（Cry1,Cry2,Tim1,Per,Clk and Cyc）showed that these genes had an important roles in the production of alate and apterous parthenogenetc female,male,gynoparae,and oviparae in offspring.Compared with the control group（injection dsGFP）,the number of apterous parthenogenetc female in treatment group（injection dsCry1,dsClk and dsCyc）was significantly decreased,and the number of male and oviparae was significantly increased.However,the number of apterous parthenogenetc female in treatment group（injection dsCry2,dsTim1 and dsPer）was significantly increased,and the number of male and oviparae was significantly decreased.And melatonin plays an important role in the photoperiodic clock（photoperiodic timer）process of aphids.