Analysis Of Gene Expression During The Larval Molting Stage In Bombyx Mori By The Whole-genome Oligonucleotide Microarray

Ecdysis is a unique biological phenomenon common to arthropods and nematodes. During the ecdysis, the old exoskeleton/cuticle is shed and replaced by a new exoskeleton. Holometabolans have four different stages in the whole life cycle including the egg, larvae, pupa and adult and generally undergo larval molting as well as metamorphosis continually. So, holometabolans are regarded as an ideal model for studying the ecdysis development. Although previous studies have confirmed that the insect ecdysis is mainly coordinated by the juvenile hormone and the molting hormone, the systematic analysis of the gene expression during larval molting and metamorphosis will promote understanding the molecule mechanism of insect ecdysis. Up to now, gene expression during metamorphosis in some holometabolous insects has already been reported, whereas the gene expressed changes during the larval molting have not been detailedly analyzed. The Bombyx mori, the representative organism for the biological studies of lepidoptera insects, belongs to the holometabolan and has irreplaceable superiority in insect evolution, development, pest control and other aspects. In 2003, the draft sequence for the genome of Bombyx mori, has been finished successfully. In 2006, the first whole-genome microarray with 22,987 70-mer oligonucleotides probes covering the presently known and predicted genes in the silkworm genome has designed and constructed. These provided us a good foundation in studying the ecdysis mechanisms at the whole-genome level. In present study, we used this ologonucleotide microarray to survey the gene expression changes during larval molting in Bombyx mori, including 10 developmental time points from fourth instar larvae to 8 hours of fifth instar. Main results are as follows:1. Summary of gene expression during larval moltingWe considered a gene to be expressed if its signal intensity exceeded a value of 800 signal intensity units after subtracting the background. Based on the statistical analysis of the microarray data, we have found that 10571 genes are expressed during the growth and ecdysis of fourth larval instar. Of 10571 expressed genes, 8687, 8364 and 9607 genes are expressed in just awaking stage, feeding stage and the molting stage, respectively. Taken together with Gene Ontology functional annotations results showed that they could classify into cell,cell part and cell organellein in cellular component aspects. At the same time, these expression genes are mainly classify into binding, catalytic activity and transportation activity in the molecular function aspects. In addition, they are mainly involed in cell process, metabolism process and the biology regulation process in the biology process aspects.2. Change tendencies of gene expression during larval moltingScatter plot analysis of gene expression in different time points during larval molting show that gene expression change is always small in four time points including 1 day, 2 day, 3 day of fourth instar and 8 hours of fifth instar, compared with the expression in 3 day of fifth instar. This is because these four time points and 3 day of fifth instar belong to feeding stage and the physiological activities in these time points are similar. However, the changes are relatively obvious in 3 day of fourth instar, indicating that some specific genes are expressed in this time points and may be associated with the onset of ecdysis. Moreover, gene expression changes during the molting stage are remarkable and are related to the differences in metabolic activity between the molting and the feeding stages. The gene expression at awaking stage, belongs to intermolting between the molting and the feeding stage, is flat.3. Hierarchical clustering of genes expressed differentially during larval moltingWe investigate the similarities and differences of gene expression according to transcription level by hierarchical clustering. The result showed that gene expression of 7572 changed at least in one time points. The hierarchical cluster analysis of differentially expressed genes revealed that the fourth instar feeding stages, fourth or fifth instar awaking stage as well as fifth feeding stage were firstly clustered into one branch and then this branch clustered together with the branch that the larval molting stage formed. The hierarchical clustering rescults indicated that there are similar physiological activities in the awaking and the feeding stage in Bombyx mori, but those are absolutely differences in the molting stage.4. The differences of gene expression during larval moltingWe identify the diffenentially expressed genes if a change between one stage and the other stage of more than two-fold. Compared the gene expression pattern between the feeding and the molting stages in fourth laval instar, we found that 484 and 720 genes are up-regulated in the feeding and in molting stages, respectively. Also, 398 and 222 genes are up-regulated in molting stages and 0 hours of fifth larval instar, respectively, comparing with the gene expression between the fourth instar molting stage and the fifth instar awaking stage. Moreover, we found that 298 and 117 genes are up-regulate expressed in 0 hours and feeding stage of fifth larval instar larva.The up-regulated genes in the feeding stage mainly contain metabolism enzymes such as peptide enzyme, amylase, carbohydrase, lipase, esterase and so on. The up-regulated expressed genes in molting stage are mainly relative to histogenesis, transport of substance, the regulation of transcription and translation, and cellular degeneration. The inositol phosphatase gene up-regulated in 0 hours of the fifth instar is involved in the digestion of textile fiber. Antenna esterase and antenna binding protein might be involved in seeking foods and distinguishing odors.5. The hormone and ubiquitin-related gene expression during larval moltingSome of stage-specific expressed genes are relative to synthesis, metabolism and signal transduction of juvenile hormone and molting hormone during larval molting. Juvenile hormone diol kinase and juvenile hormone esterase are expressed in later feeding stage and molting stage, respectively. So we speculated that they may be participated in the degeneration of juvenile hormone in different stage. Transcriptional factor BR-C Z1 is expressed in 3 day of fourth larval instar in feeding stage. This expression patter is coincided with the change of ecdysone titer, indicating that BR-C Z1 was begun to express under the inducement of ecdysone and may play a key role inonset of the larval ecdysis. More importantly, the ubiquitin proteinases are specifically expressed during the molting stage, suggesting that the ubiquitin-proteinases signalling pathway is exist in Bombyx mori and may be involved in the programmed cell death during molting stage.