Morphological Research On Extrusomes Of Four Hypotrichous Ciliates

Extrusomes are a special type of organelle that are coated by the membrane in the cortex of the protozoan cell and ejected its contents when stimulated.Different ciliates have different extrusomes which perform different functions.The morphological study of extrusomes not only helps us in better understanding the structural diversity and complexity of eukaryotic cells,but also the relationship between cell structure and function of eukaryotes,and provides a new perspective for ciliate taxonomy and systematics.Hypotrichous ciliates are highly differentiated protozoa group.However,compared with its high species diversity and the level of differentiation between groups,the understanding of their basic morphology of extrusomes is still insufficient,especially from the ultrastructure point of view.In this paper,for four ciliate speices,Bakuella subtropica,Notohymena apoaustralis,Neobakuella sp.,and Lamtostyla sp.morphological studies were performed on their extrusomes using live observation,scanning electron microscopy and transmission electron microscopy.The results are summarized as follows.Bakuella subtropica has yellow-green cortical granules which are spherical and about 0.5?m in diameter.According to the results of the ultrastructure,the granules can be ejected,usually position under the pellicle.The granules have two layer membranes,and the membranes enclose the pigment material that having high electron density which is not substructure-differentiated,and so these granules should be pigmentocysts.Pigmentocysts are formed in cytoplasmic vesicles.The outer membrane is formed first,and then gradually filled with pigment.The Pigmentocyst eventually detach from the vesicle and locate beneath the pellicle;the process may involve endoplasmic reticulum and mitochondria.After the pigment material is ejected,the organelle membrane is not fused with the pellicle and may be re-used in the cytoplasm.Notohymena apoaustralis has yellow-green cortical granules which are spherical and about 0.5?m in diameter.Transmission electron microscopy showed that the granules are two-layer membranes with high electron density pigment clusters,and the pigment clusters consist of particles of about 60 nm.They could be ejected during SEM sample preparation,so the cortical granules are pigmentocysts.Although both Notohymena apoaustralis and Bakuella subtropica have pigmentocysts,their pigment substances are differences in ultrastructure,which may be caused by different chemical components.Both pigmentocysts of these two cells originated from cytoplasmic vesicles,and it is speculated that the pigmentocysts in hypotrichous ciliate may be derived from vesicles.At the posterior area of Notohymena apoaustralis,spindle-like structures of 1-2 ?m long are found in both the vicinity of the pellicle and the deep endoplasm.The spindle-like structures have two layers of membranes,which wrap high electron density pigment material with uneven distribution;and they could be ejected during electron microscope sample preparation,so the cortical granules are extrusomes.The manner in which the organelle is only distributed in the posterior area of the cell and its absence in morphological changes is not found in other types of extrusomes,and thus the spindle-like structure represent a new type extrusome.And because of its structural similarities with mucocyst,it is temporarily named mucocyst-like extrusome.Neobakuella sp.has a large number of yellow-green cortical granules,ellipsoidal,about 0.6?m long and 0.3?m wide.Viscous liquid material can be ejected during SEM sample preparation.A lot of granules are found in the vicinity of the pellicle by transmission electron microscopy,these granules are two-layer membranes,and they can be divided into two parts according to the difference of electron density.These granules have certain similarities with the pigmentocyst and mucocyst,and they are belonging to the intermediate type of them.But because the cells have yellow-green cortical granules,so the granules are named as pigmentocyst-like extrusome.Ultrastructural studies have shown that the pigmentocyst-like extrusomes of Neobakuella sp.and the pigmentocysts of Bakuella subtropica and Notohymena apoaustralis,which are both yellow-green,but they are different types of organelles,and this phenomenon corresponds to the difference in shape under light microscopy.Therefore,it is suggested that the taxonomic weight of the cortical granules' shape can be improved in the taxonomic identification of the ciliates,that is,ciliates with different shapes of cortical granules should not be classified as the same species,and may not even be the same genus.The spherical or ellipsoid cortical granules of Lamtostyla sp.are arranged in a flower ring only with the cirri and dorsal kinety.Observation by transmission electron microscopy,these granules are single-layer membrane structures with dense electron density.This kind of granules are similar to the ampules in morphology and distribution,but no ejection phenomenon was found during the experiment,so temporarily name it ampules.Based on the phylogenetic tree that constructed by the SSU rRNA gene sequences of the four ciliates in this paper and the related hypotrichous ciliates,we find that Pseudourostyla nova,Pseudourostyla cristata and Anteholosticha monilata are clustered well,probably because they all possess trichocyst-like extrusomes.Anteholosticha antecirrata,Neobakuella sp.,Bakuella granulifera,Urostyla grandis cluster well that probably because they all possess elliptical yellow-green cortical granules,namely pigmentocyst-like extrusome.In oxytrichidae,all the groups could be divided into pigmentocyst branch and non-pigmentocyst branch except stylonychinae.These phenomena indicate that the type of extrusome is consistent with molecular phylogenetic,and this may provide clues for solving the phylogenetic problems of hypotrichous ciliates.In conclusion,this study firstly revealed the ultrastructure of the cortical granules of four hypotrichous ciliates,confirmed that the three of the granules are extrusomes,and firstly found that one cells could have two types of extrusome.It is revealed that the pigmentocyst-like extrusomes of Neobakuella sp.have the characteristics of both pigmentocysts and mucocysts;and the mucocyst-like extrusomes of Notohymena apoaustralis are different from the other types in distribution and injection process.This indicates the high diversity and complexity of organelle differentiation in different groups of hypotrichous ciliate.Further investigations show that before localization to the pellicle,the pigmentocysts,pigmentocyst-like extrusomes and mucocyst-like extrusomes have both undergone the process of development,transport and separation from vesicles in the cytoplasmic,which may be the commonality of different extrusomes.In addition,the relationship between the structure of pigmentocysts and the taxonomic status of species was summarized,and the evidence for organelle level was provided to improve the weight of pigment granules in taxonomy.