| Ecdysone is one of the most important hormones in insects, which exerts functions through heterodimer receptor consisted of both ecdysone receptor (EcR) and ultraspiracle protein (USP). Recent studies indicate that ecdysone and EcR/USP participate in the regulation of vital biological events in insects including molting, metamorphosis, neuronal plasticity, immune, reproduction, diapause, behaviour, social insects’ caste differentiation and so on. Estrogen-related receptor (ERR), an orphan nuclear receptor, is concerned with glycolytic pathway in Drosophila, and plays an improtant role in metabolic regulation and developmental processes.Polyrhachis vicina Roger is a species of typical complete heteromorphic social insects, passing through the four stages of egg, larva, pupation and adult in its lifetime. Ployrhachis vicina Roger possesses the characteristics of castes differentiation and division of labor, with a wide range of polymorphisms and highly complex nervous system.So,it is a good material to study growth, development, physiology and biochemistry of social insects.In this study, we investigated the expression location and expression level of EcR protein in Polyrhachis vicina Roger by some experimental methods including florescent real-time quantitative RT-PCR (qRT-PCR), western blot and immunohistochemical methods and so on. The relationship in protein level between EcR or USP with ERR was also explored by food-mediated RNA interference (RNAi). The results of investigations are as follows.1. The expression level of EcR protein in different developmental stages and castes was investigated by western blot. The results show that EcR protein is expressed throughout all developmental stages, and presents a specific expression pattern in different stages. During metamorphosis, the least expression level is found in eggs. The levels gradually increase from first instar larva, up to the highest in third instar larva, and decline in pupa. All expression results in the translational level roughly coincide with the results in the transcriptional level investigated previously. After adult emergence, the expression level of EcR protein in all three castes are obviously lower than third or forth instar larva, whose ranks from highest to least in turn is female, male and worker ants. The results with specific expression pattern hint that EcR maybe have certain functions in different developmental stages and castes of Polyrhachis vicina Roger.2. Immunohistochemistry method was used to anlysis the expression location and level of EcR protein in different castes’ adults of Polyrhachis vicina Roger. Results indicate that EcR protein distribute widely in many different body tissues, and the expression level showing a significant differences during different tissues or castes.In the brain, EcR protein is expressed strongly in the mushroom bodies of all three castes, especially in the calyx and Kenyon cells. The positive signals in the optic lobe, center complex and suboesophageal ganalion of male ants are obviously stronger than the worker and female ants(P<0.05). In the deutocerebrum(also known as olfactory lobe), the positive reaction in worker is strongest, and significantly higher than the other two castes(P<0.05). In tritocerebrum, the weakest positive reaction is found in female ants followed by male ants and worker ants. The difference of EcR protein expression among three castes indicate that EcR maybe take on distinct functions in different castes. According to the results of distribution and quantity of EcR in the brains, we speculate that EcR maybe play an important in the regulation on the neuronal plasticity, learning, memory and behavior activities. In the chest, EcR positive particles exsist in the thoracic ganglia of all castes, and the quantitative relation ordered from most to least in turn is as follows:worker, female and male. The castes-specific expression pattern maybe have something to do with the behavious complexity degree of different castes, and EcR in the thoracic ganglia is likely to play a key function in activities of chest muscles and foots. In the abdomen, the prominent positive reaction mainly appears in the gonad of all three castes, including oocyte of the differentition and growth stages in worker, spermatocyte, male accessory glands and seminal vesicle in male, oocyte of the differentition, growth and yolk forming stages, follicle cells and nurse cells in female. Moreover, a certain degree of positive reaction can be also observed in fat body cells located in peritoneal cavity. On the basis of the research advance reported and the distribution of EcR in the abdomen, it may be inferred that EcR may be associated with gonadal development, spermatogenesis, oogenesis and so forth, and plays an important role in growth and development of Polyrhachis vicina Roger.3. In order to explore the relationship between EcR or USP withERR in protein level, the expression of EcR and USP mRNA were knocked down by RNA interference(RNAi) technique, and the expression of involved genes are determinated by qRT-PCR, western blot and immunohistochemistry. The results are as follows.(1) Three DNA fragments with 451bp,646bp and 438bp respectively in length were amplified with the specific primers designed according to known cDNA sequence of EcR, USP and GFP, and the dsRNA of three genes used for RNAi were further synthesized with above three amplified DNA fragmants as template using T7 RiboMAX Express RNAi System. DsRNA solution of EcR and USP were diluted with 300mM sucrose solution to a concentration of 15ng/ul and 10ng/ul, the concentration of GFP dsRNA is consistent with the concentration of EcR or USP dsRNA solution.(2) The feeding method was tested for introducing EcR, USP or GFP dsRNA into the body of worker ants. The feeding was conducted once a day for continuous 12 days, and the mRNA and proteins were extracted to detect once every 3 days. The results indicate that, EcR expression in mRNA and protein levels display significant downregulation at three time point including three days, six days and twelve days after RNA inteference. USP expression in mRNA level displays significant downregulation at all monitoring points. The expression level of ERR protein appears obviously decreased at the third and twelfth day after EcR-RNAi, and obviously decreased only at the ninth day after USP-RNAi. All these results indicated either EcR or USP has a regulatory role on the expression of ERR in the protein level, which is closely related to developmental stages and interfering efficiency.(3) Immunohistochemical results show that, after EcR-RNAi, reduced obviously positive reaction of EcR mainly occur in pedunculus of mushroom body, central complex body, and deutocerebrum in the brains. Moreover, the positive expression appear also reduced to a certain extent in fat body cells located in the peritoneal cavity. The positive reaction of ERR protein significantly decreased in Kenyon cells and β-lobe of mushroom body and deutocerebrum. In addition, the amount of positive particles in the central complex body and calyx of mushroom body also has a certain degree of reduction. The results indicate the expression of ERR can be regulated by EcR with a tissue-specific way.(4) The dipping methods was tested for spraying EcR, USP or GFP dsRNA on the outer epidermis of forth instar larva and pupa. The dipping was conducted once a day for continuous 12 days, and the mRNA and protein were extracted to detect the expression levels after the RNA interference was finished. The results indicate that EcR and USP mRNA are significantly reduced in forth instar larva, whereas in the pupal stage have no significant difference.The expression levels of EcR and ERR protein in the EcR-RNAi groups have no significant different changes in statistics compared to the GFP-RNAi groups in forth instar larva, which may be caused by the lack of interfering efficiency. However, in the USP-RNAi groups, ERR protein is significantly declined compared to the GFP-RNAi groups, which indicates USP can regulate the expression of ERR in forth instar larva.This study reveals systematically the expression location and level of EcR protein for the first time. Meanwhile, we successly make use of sucrose solutions-mediated RNAi to explore the relationship between EcR or USP and ERR in the protein level, and the roles of the three genes on growth and development of Polyrhachis vicina Roger. The research results can provide a theoretical basis for the physiological and biochemical studies in Polyrhachis vicina Roger and other insects. |
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