| When insects receive the environmental signals, such as temperature, photoperiod, humidity etc., they will enter a state of programmed developmental arrest called diapause which is characterized of low metabolism, low-speed development ahead of the unfavorable environment. Insect diapause is an ideal model to study animal development, it will help us to understand the resistance of insect and supply a feasible method for pest biocontrol. Diapause could occur in different stage of life cycles, such as egg diapause, larval diapause, pupal diapause, and adult diapause. Current research result demonstrates that different diapause have different mechanism.The cotton bollworm, Helicoverpa armigera (Lepidoptera noctuidae) is a major agricultural pest which belongs to pupal diapause. It is well-known that pupal diapause is regulated by prothoracicotropic hormone (PTTP) and ecdysone. Classic theory of pupal diapause think that a shut-down of prothoracicotropic hormone (PTTH) synthesis and/or release and the subsequent failure of the prothoracic glands (PGs) to synthesize the ecdysteroids needed to promote continuous development (Denlinger, 1985). Recombinant PTTH could effectively break pupal diapause in H. armigera (Wei et al., 2005). Interestingly, diapause hormone (DH), which induces embryo diapause in Bombyx mori, was found to break pupal diapause in H. armigera (Zhang et al., 2004a) and H. virescens (Xu and Denlinger, 2003) by stimulating ecdysone synthesis and release. However, the molecular mechanism of pupal diapause termination in response of ecdysone is yet unknown. With these questions in mind, we injected ecdysone into diapausing pupae to screen the related genes from pupal diapause termination by using differential display PCR (DD-PCR). Thirty cDNA fragments were isolated from brain of diapausing pupae 24 h after injection of 20-hydroxyecdysone, and three genes were identified by homology search, cytochrome c oxidase subunit 1 of Feltia jaculifera (90%) , vacuolar ATPase subunit e of Manduca sexta (92%) and NADH dehydrogenase 1 alpha subcomplex subunit 6 Culex pipiens (70%) respectively.Cytochrome c oxidase subunit 1 is the largest subunit of COX, the terminal complex of electron transfer chain (ETC), and comprises the active core of cytochrome c oxidase with COX2 and COX3. We obtained the full length of H. armigera cox1 (Hea-cox1) cDNA which has an open reading frame of 1530 nucleotides encoding a putative protein of 510 amino acid residues, with CGA as start codon. To evaluate the response to different energy demands during pupal development and at diapause termination, we assessed the expression of Hea-cox1 mRNA and protein, COX activity and its phosphorylation. The results show that Hea-cox1 expression at the mRNA and protein levels is associated with COX activity, and high levels of Hea-cox1 expression and COX activity are present in nondiapause pupae, suggesting that low energy metabolism provided by oxidative phosphorylation in mitochondria in diapause individuals is necessary. After diapause is broken by injection of 20-hydroxyecdysone, expression of Hea-cox1 mRNA and protein increases gradually and COX activity increases significantly. Furthermore, Hea-cox1 phosphorylation is closely correlated with COX activity, suggesting that reversible protein phosphorylation might play a key role in insect diapause through suppressing the rate of energy product.NADH dehydrogenase 1 alpha subcomplex subunit 6 (ndua6) is an adhere subunit of the complex I in the electron transfer chain (ETC), encoded by the nuclear genome. In the brain of H. armigera pupae, mRNA transcript of ndua6 is higher in non-diapausing than diapausing pupae. No significant difference of ndua6 mRNA transcript was found during diapause preparation and maintemance stage. When diapause was broken by injection of 20-hydroxyecdysone, mRNA transcript increase significantly within 24 h after injection. Protein expression of the ndua6 in non-diapausing pupae is higher than that of diapausing pupae. Vo-ATPase subunit e is a subunit in V0 complex of vacuolar ATPase and is a twice trans-membrane protein. In the brain of H. armigera, the same mRNA transcript was found on pupae day 0 and day 1 between non-diapausing and diapausing pupae. With the progress of diapause, mRNA transcript of diapausing pupae has the same profile as non-diapausing pupae and after pupae day 15, transcript of diapausing is significantly low than non-diapausing pupae. mRNA transcript increase significantly within 1 day after injection of 20-hydroxyecdysone. ATPase activity of nondiapause pupae is high at the early pupal stage and decrease during pupal development. the ATPase activity in diapause type pupae show a stable low level, and increases significantly within 1 day after injection of 20-hydroxyecdysone. ATPase activity in nondiapause pupae is higher than diapausing pupae. |
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