Effects Of Prolonged Starvation On The Cytomorphology, Metablic Change And Genes Expression In Planarians Dugesia Japonica

Freshwater planarians are unique animals, exhibiting strong tolerance to prolonged starvation. Anadult planarian can still survive after several months of starvation. Except for the reduction in body size, thestarved planarians maintain the normal physiological function. The effects of prolonged starvation oncytomorphology, metablic change and genes expression in planarians Dugesia japonica has been done verylittle in and out of abroad. In this paper, the HE staining and transmission electron microscopy (TEM) wereemployed to observe the cytomorphological changes in planarians Dugesia japonica during prolongedstarvation; The automated biochemistry analyzer was used to detect some enzymes activity; Thedifferential-display RT-PCR and differential proteomics approach were used to investigate the up-regulatedgenes and proteins; The RACE technique was used to clone the full-length ofDjHSP90/DjGRP78/DjHDP40cDNA sequences; The fluorescent real time PCR was employed to detectthe changes of DjHSP90/DjGRP78/DjHDP40gene expression during prolonged starvation; Thewhole-mount immunohistochemistry was used to determine the tissue distribution of DjHSP70andDjHSP90; The colorimetry method was used to investigate the starvation-induced oxidative stress andantioxidant defenses. The results were as follows.1. Tissue structure and cytomorphological changes during prolonged starvation in planarian DugesiajaponicaIn the normal living planarian Dugesia japonica, the tissues structure is very integrity, and theboundary of different tissues is obvious. Whereas, the boundary of different tissues is obscure, and a largenumber of acidophile cells can be seen in the intestinal tissues and parenchymal tissues in starved animals.Notably, these acidophile cells undergo a process called “cell autolysis”, of which the cytoplasms aredisintegrated into numerous vesicles. The ultrastructure observation reveals that the nuclei of some cells areempty, the euchromatins are resorbed and only little heterochromains remain in the nuclei. And partial cellswith little cytoplasm, of which is full of vacuoles, are more similar to what is described “autophagic celldeath”. The selective cell death may provide nutrients for planarian survival during prolonged starvation.2. Changes of six enzymes activity and the expression level of PCNA mRNA during prolonged starvation in planarian Dugesia japonicaThe results show that the activity of alanine aminotransferase (ALT) and aspartate aminotransferase(AST) elevated apparently during starvation,10-fold higher than the control level, but reduced to thenormal level after re-fed; The eznzyme activity of creatinekinase (CK) and the lactate dehydrogenase (LDH)are very strong (250U/g protein and80U/g protein, respectively) in normal palnarians, but decreasedsignificantly after4-6weeks starvation, and returned to the normal after re-fed. The activity of alkalinephosphatase (ALP) decreased during starvation, whereas the activity of acid phosphatase (ACP) increasedapparently; In response to starvation, the activity of~140kD and~40kD proteinases increased drastically.However, no changes of the expression level of PCNA mRNA during starvation were observed, whichindicated that starvation had little influences on planarian stem cells.3. Screening the up-regulated genes and proteins induced by prolonged starvation in planarianDugesia japonica by differential-display RT-PCR and by differential proteomics approachThe up-regulated cDNA fragments induced by starvation were screened and sequenced. The resultsshow that some interesting ESTs are involved in cell metabolism, cell death, cell stress, proteinases, RNAbinding proteins, transcription factors and cytoskeleton proteins. The fluorescent real time RT-PCR wasfurther used to verify the up-regulated genes, and found that the expression levels of DjHSP70, DjHSP90,DjHSP40and DjClg3A increased after1months of starvation, but the expression level of DjGRP78remained stable.Two-dimensional electrophoresis (2-DE) was firstly used to separate the total proteins from thenormal and starved tissues of planarians, and found that over2000protein spots were separated in starvedplanarian tissues, over1000protein spots were differentially expressed. Parts of the up-regulated proteinspots were identified by mass spectrometry, and subsequently the data from mass spectrometry wereblasted in NCBI database and planarians protein database, the results show that these proteins are belongedto the groups of stress related proteins, stem cell related proteins, protein kinases, vesicle transportation,anti-oxidation proteins, receptors, transcription factors, protein degradations, cell metabolism, epigeneticmodification, cell death related protein and RNA binding protein. Of which, the over2-fold expressionlevels of proteins were HSP70/90, Piwi-1liked protein, Vasa-related protein, Nanos-related protein, Nonreceptor serine/threonine kinase, Non receptor tyrosine kinase and Receptor tyrosine kinase, Synaptosomal-associated protein, Myosin heavy chain, Peroxiredoxin-6, Cytochrome P450, Superoxidedismutase, G protein-coupled receptor kinase, HMG protein, Aminopeptidase, Peptidase U62familyprotein, Serine protease, Triosephosphate isomerase, Phosphoglycerate kinase, Caspase7,Autophagy-related protein11（ATG11）and Cysteine protease ATG4B. The above works play a goodfoundation for the further studying the function of the up-regulated genes and proteins.4. Oxidative damage and antioxidant defenses during prolonged starvation in planarian DugesiajaponicaThe effects of prolonged starvation on lipid peroxidation are measured as malondialdehyde (MDA)levels. The results show that MDA levels increased1.7-fold after30days of starvation,2-fold after60daysof starvation, and4-fold after90days of starvation. The activity of the antioxidant enzyme superoxidedismutase (SOD) increases by50%after30days of starvation,76%after60days of starvation, and123%after90days of starvation with respect to controls. The activity of CAT increases by10%after30days ofstarvation,20%after60-90days of starvation. The above results indicate that prolonged starvation leads tooxidative stress, and the activation of the antioxidant enzyme SOD and CAT can protect cells fromdamage.5. Clone and bioinformatics analysis of DjHSP90, DjGRP78and DjHSP40gene from planarianDugesia japonica.The full-length cDNA of planarian Dugesia japonica HSP90(designated DjHSP90) is2354bp,including an open reading frame (ORF) of2148bp encoding a polypeptide of715amino acids with all fiveHSP90family signatures. The ORF sequences from genomic DNA was sequenced, and found only oneintron (48bp) existed in DjHSP90gene structure. In the root of HSP90phylogenetic tree is unicellularyeast, next to plants, coelenteratas, platyhelminthes, molluscs, arthropodas, and vertebrates. Therelationships displayed in the phylogenic tree are in agreement with the concept of traditional taxonomy.The full-length cDNA of planarian Dugesia japonica GRP78(designated DjGRP78) is2121bp,including an open reading frame (ORF) of1983bp encoding a polypeptide of660amino acids with threeHSP70family signatures. DjGRP78contains signal peptides at the N-terminus and a KTEL peptide motifat the C-terminus, which suggests that it localizes in the endoplasmic reticulum (ER). In addition, the ORFsequences from genomic DNA was sequenced, and found only one intron (44bp) existed in the DjGRP78 gene structure. The GRP78phylogenetic tree shows that animals of Arthropods, Vertebrates, Cnidariansand Mollusks are clustered together, with planarians located at the root of the tree. The GRP78phylogenetic tree does not define the evolutionary relationship of species, rather it shows a current look atthe similarities between species based on the similarity of GRP78homolog sequences.The full-length cDNA of planarian Dugesia japonica HSP40(designated DjHSP40) is1378bp,including an open reading frame (ORF) of1236bp encoding a polypeptide of411amino acids with apredicted molecular mass of46.2kDa and theoretical isoelectric point of7.52. A database search with thededuced amino acid sequence indicates that DjHSP40contains four domains structure: J, G/F, CR, andC-terminal, which characteristics of DjHSP40suggestes that it belongs to the TypeⅠ HSP40subfamily.The HSP40phylogenetic tree shows that animals from Vertebrates and Arthropods are clusted together,which forms a sister branch with the animals from Platyhelminthes. The relationships displayed in theHSP40phylogenic tree are very not in agreement with that displayed in HSP90and GRP78phylogenic tree.This phenomenon suggests that stress proteins in planarians display a divergent evolution.6. Expression levels of DjHSP90, DjGRP78and DjHSP40during prolonged starvation in planarianDugesia japonicaThe fluoresent real-time quantitative RT-PCR was employed to measure the temporal expression ofDjHSP90, DjGRP78and DjHSP40during prolonged starvation in planarian Dugesia japonica. The resultsshowed that DjHSP90increased1.5-fold after30days of starvation, maintained1.6-fold after45-60daysof starvation, and reached2-fold after90days of starvation, but not restored to the normal level in there-fed (RF) animals. The transcriptional level of DjGRP78did not increase after30days of starvation, evenwas slighly lower (0.65and0.69-fold, respectively) than the control level after60-90days of starvation,and restored to the normal level in the re-fed (RF) animals. The expression level of DjHSP40mRNAincreased1.6-fold after30days of starvation, reached2-fold and2.6-fold after45-60days of starvationrespectively. After90days of starvation, DjHSP40mRNA level decreased slightly, but still higher than thecontrol level (2-fold). Other than reducing the DjHSP40mRNA level in the re-fed animals, it increased to3-fold higher than the control level.7. Expression pattern of DjHSP90and DjHSP70during prolonged starvation in planarian Dugesiajaponica The results by whole-mount immunohistochemistry reveal that the distribution of DjHSP90positivesignal is located in long lateral and parallel lines in the dorsal part of the body in1months starved animals,no positive signal is detected in control samples. The expression pattern of DjHSP90is more similar to thatof the testes-specific expression gene DeY1. The results suggest that DjHSP90-positive tissues may be thetestes tissues, and the up-regulation expression of DjHSP90may be involved in protecting testes tissuesfrom damage induced by prolonged starvation. The DjHSP70-positive cells are present throughout theplanarian body, except for the head and the pharynx. In addition, prolonged starvation does not affect thetissue distribution of DjHSP70, but can elevate its expression level evidently. Interestingly, thecharacteristics of DjHSP70-positive cells is more similar to what is described the planarian stem cells(neoblasts). It is inferred that the up-regulation of DjHSP70may have an important role in maintainingplanarian stem cell homeostasis and protecting them from starvation-induced damage.Conclusion:(1) Prolonged starvation can severely damage the tissue and cell structures in planarian, and leads toselective cell death, which may provide nutrients for planarian survival during prolonged starvation.(2) Prolonged starvation can lead to oxidative damage, which may be the major cell death inducer inplanarians.(3) To defend against starvation-induced stress in planarians, the up-regulation of HSP proteins can protectthem from damage. Especially, DjHSP70may have an important role in maintaining planarian stem cellhomeostasis and protecting them from starvation-induced death.