Function And Regulatory Mechanism Of Octopamine In Fecundity Of Nilaparvata Lugens(St?l)(Hemiptera:Delphacidae)

The brown planthopper,Nilaparvata lugens Stal(Hemiptera:Delphacidae),is a monophagous phloem-sap sucker and one of the most destructive insect pests of rice(Oryza saliva L.).When exposing to chemicals,reproductive effects of N.lugens male could be induced and transferred to its female counterpart via copulation,eliciting profound post-mating responses(PMRs)of female insects.Whereas the molecular mechanisms through which the PMRs arise from and act remain an enigma.The digital gene expression profiling(DGEP)of mated versus virgin N.lugens females at 2 DAE revealed that two components of cAMP/PKA pathway,i.e.,NlAC9 and NIPKAR were significantly up-regulated post copulation and they proved to be critical players in egg-laying and population growth of N.lugens female.Intriguingly,octopamine(OA)level in the female ovary also elevated substantially after mating.OA,the invertebrate biogenic amine analogue of mammalian noradrenaline,exerts its function as a neurotransmitter,neurohormone and neuromodulator that regulate various behavioral and physiological processes including mating,ovulation and oviposition in insects.Herein,it is proposed that OA responds to mating and modulates N.lugens female fecundity via cAMP/PKA pathway.The findings are detailed as follows:1.OA in response to mating induces the cAMP/PKA pathwayThe E1ISA assay showed that the OA accumulated much more in the ovary of mated females than that of virgin ones,similarly,the T?H,AC and PKA enzymatic activities as well as OA and cAMP level also increased significantly post copulation.Consistently,the expressions of NIT?H and NlOct?2R of females were up-regulated after mating and primary components of cAMP signaling pathway such as NlAC9,NIPKAR,NlCREB1 and NlCBP were also induced by copulation;meanwhile the p-CREB1 intensity elevated.On the other hand,exogenous injection of OA partially stimulated levels of cAMP,AC,PKA and p-CREB1 along with transcript levels of NlOct?2R,NIAC9,NlPKAR,NlPKAc and NlCREB1.These findings preliminarily demonstrated that OA responded to mating and initiated cAMP/PKA signaling pathway.2.The functions of OA in N.lugens fecundity(1)All target genes involved in OA/cAMP/PKA signaling pathway were predominantly transcribed in female adults post emergence and exhibited relatively high transcript levels in the brain,fat body and ovarian tissues,suggesting the critical role of OA in fat body and ovary via cAMP/PKA pathway.(2)The effects of NIT?H silencing on female female physiology,biochemistry and fecundity:Compared with the dsGFP-treated group,knockdown of NlT?H significantly diminished transcript levels of NlTvH,NlOct?2R and OA content while enhanced TA level.In addition,the expressions of cAMP/PKA components and p-CREB1 level sharply reduced,paralleled the cAMP level,AC and PKA enzymatic activities decreased;the physiological and biochemical parameters including body weight,fat body and ovarian soluble protein content;NlJHAMT,NlMet,Vg,VgR expressions;JH ? titer and Vg protein level significantly dropped.Moreover,silencing NlT?H severely disrupted female egg laying,oviposition period and longevity.JH topical application partially rescued NlT?H,NlJHAMT,NlMet,Vg expressions,JH ? titer and Vg protein level in the dsT(3H+methoprene group relative to the control(dsT?H+acetone).On the other hand,dsT?H+OA treatments restored the NlT?H,Vg,VgR expressions,Vg protein level,OA content,and JH ? titer as compared to the dsT?H+PBS group,manifesting that NlT?H implicates in female fecundity by modulation of OA and JH synthesis and the synergistic interaction between them.(3)Virgin+OA treatments induced the expressions of NlJHAMT,NlMet and JH ? titer compared with virgin+PBS control.Compared with the virgin+acetone control,NlT?H transcribed level and OA content increased significantly in the virgin+methoprene group.These findings indicated a collaborative interplay between OA JH in that OA partakes JH synthesis and vice-versa.(4)Relative to the dsGFP treatment,the ovarian development was strikingly impaired and retarded with stunted morphology,fewer ovarioles and mature eggs.Moreover,scarce Vg uptake by oocytes and no conspicuous patency were visualized between nuclei by IF staining post RNAi-treatments Additionally,the number of progenies,hatching rate and PGI in the F1 dramatically declined in RNAi-silenced groups.These observations strongly pointed to the fact that OA coupled with the cAMP/PKA pathway operated in concert with JH to modulate physiological,biochemical and reproductive aspects of N.lugens.Overall,this study(1)characterized the pattern of OA responsive to copulation;(2)elucidated the physiological role of OA;(3)highlighted the synergistic effects on the regulation of N.lugens fecundity exerted by OA and JH.