The Expression And Function Analysis Of Several Enzymes Which Are Related To Insects Molting

Insect molting is a special life phenomenon of nematodes and arthropods during metamorphosis and is initiated by the molting hormone,20-hydroxyecdysone (20E). Molting includes 1) the activation of dermal cells,2) separation of the epidermal cells,3) secretion of molting fluid,4) formation of the new epidermis, and 5) shedding of the old epidermis. During the molting process, the associated enzyme expression undergoes significant changes. In this study, we analyzed the expression and function of enzymes that are related to the insect molting hormone in the silkworm Bombyx mori and the mirid bug Lygus hesperus. The results showed a clear relationship between molting hormone and its related enzymes during the molting process, in particular phenol oxidase, dopa decarboxylase and chitinase. The main findings are as follows:(1) The polyphenoloxidases, BmPPO1 and BmPPO2 were cloned and identified from the silkworm Bombyx mori L. The BmPPO1 cDNA contains an ORF of 2058 bp and the deduced protein sequence contains 685 amino acid residues, with an isoelectric point and molecular weight of 6.25 and 79 kDa, respectively. The BmPPO2 cDNA contains an ORF of 2082 bp and the deduced protein sequence contains 693 amino acid residues, with an isoelectric point and molecular weight of 5.6 and 80 kDa, respectively. Quantitative expression analysis of the PPOs in different stages and tissues revealed expression of the two PPOs in early stage silkworms is consistent with the molting process. The expression peak of the two PPOs is higher during the molting stage, and decreases after molting. During the early fifth instar, PPOs expression is highly variable in the head and haemolymph, while changes are negligible in the epidermis and reproductive organs. A significant increase in ecdysone levels was observed in the haemolymph of silkworm larvae artificially fed with 20E. PPO1 expression was consistently and significantly elevated compared to the control. Therefore, we concluded that PPOs expression is regulated by the molting hormone, and that it is a necessary enzyme for silkworm molting. RNA interference of PPO1 caused an obvious decline in expression and generated an incomplete pupation phenotype.(2) Dopa decarboxylase (BmDdc) was cloned and characterized from Bombyx mori. The BmDdc cDNA contains an ORF of 1437 bp and the deduced protein sequence contains 478 amino acid residues, with an isoelectric point and molecular weight of 5.8 and 54 kDa, respectively. The BmDdc expression level was very low prior to the second instar, but increased during the second sleep period, with a regular expression peak appearing in late stage (3rd instar), this peak, however, was still collelated with the sleep period. Fluctulation in Bm Ddc expression levels were largely restricted to the epidermis, head, and reproductive organs during late stage silkworm, and relatively high expression levels were detected in mature silkworms and pre-pupa. A significant increase in ecdysone levels was observed in the haemolymph of silkworm larvae treated artificially with 20E.,BmDdc expression was consistently and significantly elevated compared to the control. Therefore, we concluded that BmDdc expression is regulated by the molting hormone, and that it is a necessary enzyme for silkworm molting. RNAi-mediated knockdown of Bm Ddc in silkworm larvae, caused a significant decrease in BmDdc expression at 24 hr after injection, and a significant abnormal pupal phenotype was observed in 34.4% of the pupae. BmDdc activity was detected by liquid chromatography, but there were no significant changes in 5th instar. To examine the intracellular localization of BmDdc,we generated a polyconal antibody, recombinant BmDdc generated using a bacteria expression system. Following purification,the antibody was used to detect BmDdc expression in the cytoplasm near the plasma membrane.Using the normal secretory pathway encompassing the endoplasmic reticulum and Golgi, the enzyme is transported out of the cell to function in epidermis hardening and the tanning process. We speculate that significant portation BmDdc synthesis occurs in the cells prior to the molt, and is then released extracellularly after the molt. Using tissue immunofluorescence, we found that the protein was mainly localized on the surface and in the nerves of the head and epidermis, which is consistent with its role in the molting process.(3) As there are not many references available on the mirid bug Lygus hesperus, the study of the molting mechanism in this insect was based on similarities with Bombyx mori and other insect. We determined the molting hormone titers of L. hesperus, and found a high degree of consistency in the titers of both male and female insects. Titers increased during the late fifth instar and then decreased after molting, which is consistent with the role of 20E seen in other insects. We cloned the ORFs of several enzymes that may be related to the molting process, and examined their expression profiles in different stages and tissues. Our research showed that a chitinase gene exhibited a consistent periodicity that was synchronized with the molting process. Early induction (newly molted 5th instar nymphs) of the molting hormone significantly increased insect mortality, in particular in the nymphal stage, but also significantly reduced the probability of nympahl metamorphosis, and delayed expression of the chitinase gene. Late induction (6th day of 5th instar:note-5th instar last about 12 days at 16.9℃) of the molting hormone also increased insect mortality, but the effects became more obvious in the adult stage. The latter also significantly reduced the probability of metamorphosis. Furthermore, adding ecdysone to 5th instar extended the molting process and resulted in>20% mortality during metamorphosis. This indicates that a high concentration of molting hormone blocks L. hesperus metamorphosis, which is contradictory to the role of molting hormone in other insects.Researches into the mechanism driving insect molting is critical for understanding insect biology, has potentical to facilitate the development of novel methods for control pest species and conversely,has the same potential to improve economically viable insects. The regulation of the molting hormone on the molting process can be applicable to actual production. For example, when the molting hormone was applied at the end of 5th instar Bombyx mori, the silkworm duration was shortened, aging and metamorphosis were promoted, and the cocooning process was advanced. In the mirid bug Lygus hesperus, ecdysone blocked its metamorphosis, which maybe useful for future pest control methods. Therefore clarifying the relationship between insect molting and associated gene expression and regulation, can provide new ideas for pest control, and may promote new usage of currently available biological resources.