Morphology Of Vegetative Cells And Resting Cysts And Identification Of Encystation Genes In Oxytricha Trifallax

As highly differentiated unicellular eukaryote,many ciliates form resting cysts under adverse environmental stresses,and excystment occurs when conditions are suitable.A series of physiological and morphological changes happen during the encystation,which are the life phenomenon regulated by multiple genes.Study on encystment of ciliates not only helps in understanding how eukaryotes resist adversity in the material synthesis,metabolism and signal transduction,but also provides new information for exploring the structure and function,and origin and evolution of eukaryotic cells.Although there are some morphological studies on the ciliates during encystment,the dedifferentiation of cortical ciliature is less understood.And molecular mechanism for encystment in ciliates is scarely known due to limited investigation.In this study,we explored morphological changes in cytoplasmic organelles and continuous differentiation of cortical ciliature,encystation genes and signaling pathways of Oxytricha trifallax during encystment by electron microscopy and transcriptome technology.1.Morphology of vegetative cells and resting cysts in O.trifallaxCell size was 55 μm × 24 μm;there were three frontal,one buccal,four frontoventral,three postoral ventral,two pretransverse ventral,and five transverse cirri,six dorsal kineties.During encystment,the undulating membranes,oral cavity and adoral membranelles were first absorbed;then V/3,V/4,and the 6 or 7 marginal cirri located in the middle of the cells were contracted into the cytoplasm forming short cirri;IV/2,V/2,VI/2,and the rest of the marginal cirri disappeared in turn;the frontal and frontoventral cirri were gathered and then disintegrated as the cells became spherical;short cirri were absorbed finally.In contrast to dedifferentiation of the ventral ciliature,the naked basal bodies of dorsal bristle units re-differentiated.The cyst of O.trifallax was about 15μm in diameter and covered with a typical four-layer cyst wall wrinkled on the surface.The cytoplasm contained a large number of autophagy,mitochondria,one macronuclear nodule fused by two,and one or more spherical micronucleus.The results showed that:(1)O.trifallax formed kinetosome-resorbing cyst during encystment.The ventral ciliature dedifferentiated in order,while the dorsal bristle units re-differentiated.(2)Comparing the encystment process of O.trifallax and the reports of other ciliates,it was concluded that the dedifferentiation pattern of the ventral ciliature may vary from species,but the closer the genetic relationship was,the more similar the dedifferentiation pattern was.(3)The re-differentiation of the dorsal bristle units in O.trifallax was similar to that of Allotricha curdsi,which suggest a common feature of oxytrichines.(4)The dedifferentiation of ciliature most began in the oral apparatus in hypotrichous ciliates,probably because ciliates stop eating during encystment.2.Identification of encystation genes in O.trifallaxThe transcriptome technique was used to study the gene expression status of vegetative cells and resting cysts in O.trifallax.The transcriptome data showed that:(1)809 differentially expressed transcripts were detected.511 m RNAs were up-regulated and 298 m RNAs were down-regulated in resting cysts compared to vegetative cells.(2)Gene ontology of these differentially expressed m RNAs was performed.586 m RNAs were annotated to 1337 GO items: 286 up-regulated expressions m RNAs were consigned to 963 GO items,and 200 down-regulated expressions were matched to 516 GO comment entries.(3)These differentially expressed m RNAs are mainly concentrated in three major categories: biological processes related to carbohydrates,lipid and protein metabolism,cell division,lysosomes,extracellular space;cell components of the cell wall,and phosphokinase,oxidation;molecular functions of enzymes such as phosphokinase,oxidase,peptidase and superoxide dismutase.(4)These differentially expressed m RNAs was mapped to 207 pathways.Compared to vegetative cells,up-regulated m RNAs in cysts were annotated to 144 pathways,and down-regulated m RNAs was annotated to 180 pathways.Significantly different signaling pathways were lysosomes,c AMP,arachidonic acid metabolism,apoptosis,tricarboxylic acid cycle,autophagy,glutathione metabolism,antigens processing and presentation,sphingolipid synthesis,fatty acid elongation,and amino acid synthesis.The results showed that:(1)consistent with the increase of intracellular c AMP levels in Colpoda cucullus and Acanthamoeba during the encystation,the expression of calmodulin kinase and Na+-K+-ATPase α subunit in the c AMP pathway were up-regulated in the cysts of O.trifallax,which may prove that the c AMP pathway was involved in the regulation of dormancy in protozoa.(2)At the same time,the cells preferentially expressed a large number of HSP70 proteins in adverse environment such as starvation and overcrowding,to promote the level of endogenous oxidases such as glutathione peroxidase,superoxide dismutase and SDR family reductase,activating stress-response mechanisms to reduce environmental stress on cells.(3)In addition,corresponding to a large number of autophagic vacuoles appeared in the cysts under transmission electron microscope,differential expression of cathepsin D,L,and B in the autophagy and lysosomal pathways was also screened using transcriptome technology,which may be related to protein degradation,cyst wall formation,and the availability of nutrients during encystment.(4)The differentially expressed genes of O.trifallax were significantly enriched in autophagy and lysosomal pathways,but this phenomenon had not been reported in the study of Pseudourostyla cristata,which may be due to the fact that ciliates of the "kinetosome-resorbing cyst" were more pronounced in autophagy than ciliates of the "urostyla type cyst".Encystment of O.trifallax is a stable and orderly differentiation process,which is regulated by these different expressed genes and signaling pathways.In this study,the differentiation order and characteristics of the infraciliature in O.trifallax during encystment were summarized in detail,which can provide relevant materials for the study of the cellular structure differentiation of ciliates under different physiological conditions.On the basis of morphology investigation,transcriptomic technology has also been used to explore encystation genes of kinetosome-resorbing cyst,which can enrich the molecular-level data on encystment mechanism of different cyst-forming types among ciliates.