The Effecxs And Mechanism Of Action Of High Hydrostatic Pressure On Drosophila Melanogaster

High hydrostatic pressure(HHP) is a parameter characteristic of the Biosphere considering the volumes occupied respectively by its major terrestrial (land) and aquatic components. The oceans, which cover approximately 70% of the surface of the Earth, have an average depth of 3800 m and consequently an average pressure of 381 atm (38.5 MPa). With the discovery of piezophilic and thermophilic microorganisms, it becomes a new focus to research the potential of life's origin in the high pressure and the high temperature conditions.In addition, the Drosophila melanogaster is a typical representative as the model organism, of which the genetic backgrounds were well investigated. The genome sequencings have been completed, and some genes of Drosophila melanogaster have a high homology with higher organisms. Therefore, it is of great significance to study the mechanisms of high pressure on Drosophila melanogaster and to further explore the roles of high pressure on major life phenomena such as the origin and evolution of life.In this work, we study the effect of high pressure on the growth and development, physiological properties, and phenotypes of Drosophila melanogaster for the first time. Our results indicate that survival rate of Drosophila melanogaster reduce with pressure increases. The survival rate is zero when pressure reaches 90 MPa or more. The high pressure also resulted in increasing in the ratios of female to male and enhance in the fecundity of female. Normally, the chorionic appendages of Drosophila melanogaster are complete and regular but it is abnormal after the high pressure-treatment. They were frequently shorter and malformed, and irregularly attached to the yolk membrane. Before the high pressure-treatment the yolk membrane of Drosophila melanogaster is smooth, but the abnormal bubble or white flocculent materials appear after the treatment, and the yolk membrane appeared abnormal processes when pressure reaches 100 MPa. The untreated larvae of Drosophila melanogaster were small and white, while the larvae by high pressure-treatment were large and red. Corresponding, the pupae without high pressure treatment were small and light brown, whereas the treated pupae were large and red. The Drosophila melanogaster that survived by high pressure-treatment showed abnormalities, their adults appeared aberrant mainly in wing phenotype. Normally, the shape of Drosophila melanogaster wing is oval and the length is 2 times than that of the abdomen. It has five longitudinal veins and two crossveins as well as well established wing margins. The Drosophila melanogaster induced by high pressure-treatment showed wings degradation and obvious modifications in wing size, shape, vein patterning, wing-margin formation, and the loss of the ability to fly.Pairs of Drosophila melanogaster with abnormal wing were isolated randomly from populations after exposure to high hydrostatic pressure at 60 MPa for 20 min. These mutants retained for at least four generations. We used DNA sequencing, DNA microarray and RT-PCR technology to analyze mutagenesis mechanism.Firstly, we employed DNA sequencing to study two major genes, vg and sd, both of which involved in the wings'development. Our sequencing results indicate that sd gene does not change, but there are a total of 4 point mutations and an insertion sequence in vg gene. The first point mutation was a transversion from T to C at nucleotide 4 in exon 1, resulting in codon exchange from CTC to TTC, which was a synonymous mutation. There are a total of 3 point mutations in exon 3, two of which are transversion from G into A at nucleotide 137 and from C into T at nucleotide 332, respectively, and these two mutations are also synonymous mutations. However, the fourth point mutation was a transversion from T into C at nucleotide 96, resulting in codon exchange from TCG to CCG, which results in amino acids sequence change from serine (S) into proline (P), thus it was a misssense mutation. And the DNA sequence analysis from the vg gene revealed an insertion sequence with 17 nucleotides (5/-TAGCTGCATTAAATCTT-3/) between the first and second exon of vg, which located downstream of enhancer in intron 2. These changes of DNA sequence described above retained continuous and stable for at least four generations.Secondly, to further investigate from gene-expression level mutagenesis mechanism connected with wings mutation response to high pressure, we employed DNA microarrays to analyze genome-wide gene-expression differences between the mutants and the controls. The results showed that there are a total of 285 differentially expressed genes in the mutant Drosophila melanogaster compared with control untreated, which the difference is at least twofold (P < 0.05), 119 were up-regulated and 166 were down-regulated. To verify the accuracy of microarray data, we selected randomly 2 genes involved in wing development to perform RT-PCR. Consistency was observed between microarray and RT-PCR. Thus, results of microarray were reliable and correct. To predict functions of these 285 genes, a detailed GO analysis was conducted. GO is divided into three disjoint term hierarchies: the cellular component, the molecular function, and the biological process. These genes could be assigned to a total number of 203 different terms for'biological process', 149 for'cellular component'and 200 for'molecular function'. Metabolic process,cellular proces,biological regulation, multicellular organismal process localization, regulation of biological process, developmental process, establishment of localization, and response to stimulus within'biological process'; extracellular region, cell, macromolecular complex, organelle, organelle part, and cell part within'cellular component'; catalytic activity, structural molecule activity, transporter activity, binding, transcription regulator activity, and molecular transducer activity within'molecular function'. The cell signaling pathways regulate important physiological processes, such as cell proliferation, differentiation, growth, and development by different mechanisms. Our experimental results showed that high pressure changed a lot of signaling pathways of Drosophila melanogaster, two of which, Wnt and Notch played direct role in wing development of Drosophila melanogaster. Notch signaling is mediated by the down-regulation expression of Numb, and Wnt signaling is mediated by the down-regulation expression of Roc1a. In a word, the wing mutations of Drosophila melanogaster are the results from a combination of variation at multiple levels, including DNA sequences, gene expression and signaling pathways which induced by high pressure treatment.In this paper, we have studied systematically the effect of high pressure on the growth and development, physiological properties, and phenotypes of Drosophila melanogaster. This has important significance to further investigate the origin and evolution of life, the pressure limit of higher organisms, the organism's distribution within the earth and marine, and reached the living creatures from other planet or galaxy.