The Regulation Of Dopa Decarboxylase (DDC) On The Growth And Development Of P. Striata And Its Association With Ecdysone Receptor (EcR) And Estrogen-related Receptor (ERR)

As one of the most important enzyme in biosynthesis pathway of catecholamine(CAs),dopa decarboxylase(DDC)can catalyze L-3,4-dihydroxy phenylalanine(L-dopa)and L-5-hydroxy tryptophan to synthesize two important neurotransmitters dopamine and serotonin,respectively.Dopamine and serotonin play important roles in vertebrates and invertebrates reproduction,development,behavior,and the immune response.DDC is related to the darkening effect,and participates in ecdysis process in insects.In addition,the DDC is also involved in insect body color formation,development and regulation of lifespan and other physiological activities.Ecdysone receptor(EcR)is only known nuclear receptor ligands,whose Ligand is ecdysteroid hormones(EH).EcR and ultra valve protein(ultraspiracle protein,USP)constitute heterologous dimers EcR/USP,then combine with EH to form a ligand receptor complexes,which start cascade reaction process,and control insect development,abnormal and breeding and other life activities.Estrogen-receptor related receptor(ERR)is an orphan nuclear receptors,which can participate in estrogen signaling pathways,play a role in disease diagnosis and control energy metabolism.The previous studies in our laboratory have shown the correlation between EcR and ERR in Polyrhachis vicina Roger and suggested that DDC may correlate with EcR and ERR,but it has not yet been confirmed.Polyrhachis vicina Roger affiliate to the Class Insecta,Hymenoptera,Formicidae,which is a typical social insect,adults ant have pleomorphism and complex social behavior.Because of those reasons,P.vicina may be an suitable material for investigatigation insect growth,development and behaviors.In this study PvDDC mRNA and protein levels expression of P.vicina were investigated in different development stages,the different castes of adult by in situ hybridization,Western blot and immunohistochemistry technique.Futhermore,the RNA interference(RNAi)technology was used to silence the PvDDC gene expression,the phenotypic changes of the ants before and after interference were observed and statistics.The correlation of PvDDC,PvECR and PvERR in mRNA and protein expression levels were explored by the qRT-PCR,in situ hybridization,Western blot and immunohistoche mical technique.The results are as follows:1.The PvDDC mRNA abdominal positioning of P.vicina different caste adult were investigated by in situ hybridization technology.The results shown that PvDDC mRNA distributed widely in the abdomen,with different expression areas in the female,male and worker ants.For the worker,the strongest positive reactions were found in spermatheca and abdominal fat bodies,and followed by reproductive cavity edge and internal particles.For the male,The strongest positive reactions were found in abdominal testis,and followed by storage seminal vesicle,ejaculatory duct,fat body and spermatocyte,and weak positive reactions were found in the accessory gland.For the female,stronger positive reactions were found in oocytes,oogenesis differentiation stage,oocytes of yolk formation and fat-body.According to above mentioned results we presume that PvDDC may regulate P.vicina development of reproductive organs and physiological processes such as spermatogenesis and oogenesis.2.PvDDC protein expression of P.vicina in the different development stages and different caste adult body were explored by Western blot technology.The results shown that there were significant differences of PvDDC protein expression existed in the different development stage and caste adults.From eggs to third instars,the PvDDC protein relative expression levels appeared in a gradual upward trend,descend slightly in 4th instars which was lower than 3rd instars,and pupal stage continue to be reduced.After molting,P.vicina differentiate into three different caste including female,male,and worker..Among them,the highest PvDDC protein expressed in the male,followed by the worker,and the least in the female.In those caste adults,the PvDDC protein relative expression levels in the whole body of male was the highest,the worker's was lower than the male,but was still higher than female's and other developmental stage of the ants.We speculated that PvDDC may regulate P.vicina development in specific development period,and PvDDC protein expression differences in different caste adults may be associated with caste differentiation.3.The PvDDC protein positioning was investigated in different caste adult brain and abdomen of P.vicina by immunohistochemical technology.The results shown that PvDDC proteins have a widely positive response distributions in different caste adults brain.The mushroom bodies of different caste adult brain shown the strongest positive reactions.Moreover,for the worker,optic lobe of the brain,beta lobe,pons body and suboesophageal ganglion revealed a strong positive reactions.For the male,optic lobe of the brain and optic ganglion layer shown a strong positive reactions.For the female,lateral forebrain of brain,pons body,central complex,alpha lobe,beta lobe,the midbrain and pharynx side body exhibited a strong positive reactions.We speculate that the PvDDC may participate in the development of the nervous system,learning and memory,and complex social behavior in P.vicina.The results of PvDDC protein positioning in different caste adult abdomen was similar to PvDDC mRNA levels,which indicates that at the levels of transcription and translation,PvDDC has no significant difference in regulating abdominal development of P.vicina.4.The PvDDC expression knock down was observed and statistics in different caste adults bodys of P.vicina by RNAi silence technology mediated with feeding and infiltration.,The results shown that after PvDDC-RNAi,the ant body weight,body length and head width were decreased,growth and development of larvae was seriously impeded pupa ecdysis was delayed or died after molt,adult abdomen expands and wing dysplasia.Those shown that PvDDC has a direct effect on growth and development of P.vicina.5.The correlation of PvDDC,PvECR and PvERR on the levels of mRNA and protein were explored by the qRT-PCR,in situ hybridization,Western blot and immunohistochemical technique in different caste adults.The results shown that when the PvDDC was effectively silenced,the PvEcR expression were significantly lower in different development stages of the ant bodies,and at the statistical level has a very significant difference(P<0.01)in different groups.PvDDC effecting on expression quantity of PvERR was lower than PvEcR,but still changed obviously in a specific development stages.Among them,the expression of PvERR has declined obviously in 3rd instar larvae,pupa and female,at the statistical level reached extremely significant difference(P<0.01).The expression of PvERR reached significant difference(P<0.05)in different male groups,but there is no significant change of PvERR expression in pupa and worker.The above results shown that PvDDC associated with PvEcR is more than PvERR.We speculate that PvDDC indirect influence PvERR expression becuse of influnced by effect of PvEcR expression.These three genes work together to regulate the growth and different development period and caste differentiation in P.vicina.The PvDDC protein quantitative and positioning were explored firstly in holometamorphosis insects P.vicina in this study,and PvDDC regulating the growth and development of P.vicina is verified.PvDDC gene was effectively silenced by RNAi technology,the results exhibited that PvDDC gene may directly influence the growth and development of P.vicina,and confirm a correlation existing in of PvDDC,PvEcR and PvERR from mRNA and protein expression levels changes as well as the phenotypic changes of the ants.Those workers not only establish foundations for research of regulatory mechanism of P.vicina caste differentiation,but also provide theoretical basis for exploring growth and development regulation of other insects.