| The molecular mechanism of growth and development of two beetles, Epicauta Chinensis and Tribolium castaneum, were investigated by comparative proteomics and enzymology separately.MALDI-TOF/TOF-MS analysis of proteomics from 1st to 5th instars larvae of E. chinensis revealed that 42 proteins were differentially expressed among different stages. Our analysis revealed dynamics regulation of the following proteins:dihydrolipoyl dehydrogenase (DLD), vitellogenin (Vg), lysozyme C (Lmz-S) and Axin-like protein pry-1 (APR-1). Vg is the main nutrient source of eggs and larvae of E. chinensis. It has immunocompetence and exists in larvae from the 1st to 4th instar, but is not found in 5th instar. And its biosynthesis could be regulated by JH and food factors. DLD catalyzes the pyruvate into acetyl-CoA, the latter is the starting material of juvenile hormone (JH) biosynthesis through the MVA pathway in insects. So DLD may affect the synthesis of JH and Vg indirectly.2-DE results show that the activity of DLD in the 1st and 2nd instar is lower than the 3rd,4th and 5th instar larvae. The activity of lysozyme C is an indicator of the immunity and expressed less in the 1st instar. APR-1 negatively regulates the Wnt signaling pathway. Its overexpression could result in embryo, leg, eye and ovary ectopica or malformation. APR-1 reached the highest level in the 2nd instar, then down-regulated to the lowest level. Further investigation of five differentially-expressed proteins at mRNA level showed that only Hsc70 displayed correlation between mRNA and protein levels.The comparative proteomics in the midgut, as well as whole body without midguts and wings from 0-day,6-day and 12-day old of adult males after eclosion were carried out. Sixty-four differentially expressed proteins were successfully identified which containing some important proteins regulating the insect growth and development. Among these proteins, three digestive enzymes:glucosaccharase, carboxypeptidase D precursor and neutral alpha glycosidase enzymes ab precursor, which believed to function in digestion and absorption processes were mainly found in midguts. Their activities were very low in midguts of 0-day old adult males. Then increased significantly with their growth and tilled the peak in 12-day old adult males. There were 10 antioxidant and detoxifying related proteins, such as thioredoxin reductase, peroxidase, glutathion peroxidase, electron transfer flavoprotein a subunit, metallothionein and ethanol dehydrogenase, and so on. three proteins including immunomodulatory factor, polyphenol oxidase zymogen and prohibitin involving in immunologic defence, their amounts in 6-day and 12-day old were larger than 0-day old adults. Epidermal growth factor receptor participates in the growth and development of E. chinensis.3-oxygen acyl-acyl carrier protein reductase takes part in the biosynthesis of fatty acid. These two proteins’ quantities were the lowest in 0-day old adults, then arrived at the highest level in 6-day old adults. Corpora allata inhibin precursor restrains synthesis of JH. It upregulated along with the growth of E. chinensis adults. Ten differentially-expressed proteins and their genes were analyzed at mRNA and protein levels. Only three proteins showed correlations at two levels. This result revealed the complexity in the linkage of translational and transcriptional levels.Two sub-lethal doses (LC10, LC25) of ethyl formate (EtF) were employed to treat T. castaneum adults for 12h,24h,48h,72h, and 96h, respectively. The activities of five kinds of metabolic enzymes, the cytochrome P450 monooxygenase (P450), carboxylesterase (CarE), acetyl cholinesterase (AChE), glutathione transferase (GST), and alkaline phosphatase (ALP), as well as the transcriptional expression of these genes and heat shock protein genes were determined. The results are as follows:The activities of P450 and AChE were significantly elevated in 72h by LC10 dose exposure, but were depressed in LC25 dose treatment. CarE activity was significantly enhanced in 48h by LC10 dose exposure, however, it was reduced in 48h by LC25 dose exposure and recovered at 72h, then increased at 96h. Augment of GST activities were observed in both treatments at 12-72h. No significant changes of ALP activity were monitored in both treatments. The transcriptional levels of CYP4BN1, AChE1 and GstD2 were significantly up-regulated in two treatments indicating their involvement in the metabolism of EtF. They may participate in the metabolism of EtF. Increasement of est2 mRNAs abundance of was detected in LC10 dose treatment, however, its abundance decreased in LC25 dose treatment. Both treatments increased the expression level of Hsp68 by 48h exposure. The expression of Hsp27 was down-regulated before 48h exposure, however, it was up-regulated after 48h exposure. These two Hsp genes might play individual role in response to different doses and exposure time of EtF.The adults were exposed to high (42-51℃) and low (4℃) temperatures, and the activities of detoxifying enzymes, P450, CarE, AChE, and GST, and the expression levels of these detoxifying enzyme and Hsp genes under these stresses were investigated. The thermal and chill exposures significantly inhibited the activities of detoxifying enzymes, P450, CarE, AChE, and GST. The expression of CYP4BN1 decreased after exposed to high temperature, but increased by low temperature exposure. The expression of est2 was induced by 51℃ and 4℃ treatments. The Hsp68 and Hsp27 mRNAs were enhanced by high temperature exposure, however, the Hsp68 was up-regulated but the Hsp27 was down-regulated by short-term chill exposure. Our results indicated that CYP4BN1, est2, and Hsp68 may play positive roles in response to chill stress, while Hsp68, Hsp27, and est2 may be involved in helping this beetle enduring high temperature.In this work, the proteomic differences in E. chinensis of 1st to 5th-instar larvae, different age of fresh adults, and adult midgut were conducted using proteomic techniques. The two-dimensional electrophoresis map database of E. chinensis larvae and adults was established. The functions of differential proteins were analyzed and their expressions at the transcriptional level were studied. The molecular mechanisms of cumulative effects and transient expression of the proteins and RNAs in vivo were preliminary understood. This work would do some groundwork for the future research of diapause and the biosynthesis of cantharidin in this beetle at the molecular level.We also systematically studied the influences of sub-lethal dose of EtF and temperature stress on the growth and development of T. castaneum, investigated the physiological and molecular mechanism of lethal effect caused by adversity stress. This work will provide theoretical direction for efficiently control the grain storage pests deeply studying the mechanism of stress on insects’ growth and develonment... |
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