Morphological Study On Cells Of Cryptocaryon Irritans(Ciliophora:Prostomatea),A Parasitic Ciliate Of Marine Fishes

Cryptocaryon irritans Brown,1951 is a facultatively parasitic ciliate that is capable of infecting all of the marine teleosts causing "cryptocaryoniasis".This disease has a great harm to the aquaculture industry.In recent years,more knowledges and understanding about the basic biological characteristics and pathogeny features of C.irritans itself are valued for widely again.C.irritans has a complicated life history and can be divided into four stages:1.Trophont stage:the ciliate parasitizing and feeding inside the host fish tissues;2.Protomont stage:the transitory swimming and creeping period in water after trophont detaching from the host;3.Tomont stage:the ciliate forms tomont and performs palintomic division in tomont,yielding numerous tomites;4.Theront stage:theronts hatch out and escape from tomont,and then swim freely and achieve a secondary infection to fish.It is necessary to get a further comprehensive study about the structure of multiple organelles of C.irritans as well as their various features in different life history stages.It is not very clear about the organelles,their functions and related cytological mechanism involved in some key life stages and important life activities.In addition,C.irritans have been certained belonging to class Prostomatea in previous studies,but it is controversial about the further phylogenetic position of C.irritans inside class Prostomatea,and lack of enough morphological evidence.Propagation was conducted in this study using Larimichthys crocea as the host fish,and cells at each stage of the life history of C.irritans were collected precisely.Using observation in vivo,silver staining,fluorescence labeling techniques,scanning and transmission electron microscopy and enzyme cytochemistry,detailed morphological observation was conducted from both microstructure and ultrastructure level,and molecular phylogenetic analysis was conducted by 18S rRNA gene sequencing and construction of phylogenetic tree.An overall and systematically description of many organelles and structures were conducted,and many features about structure and life activity phenomena were revealed,Phylogenetic position of C.irritans was made clear and definition of morphology description of C.irritans was listed out.This study is useful to provide new data further perfecting and illustrating the cell morphology and basic biology features of C.irritans and even other fish parasitic ciliates.The main findings are as follows:1 Organelles and strutures in oral region of different life history stages of C.irritansExcept trophonts and protomonts of C.irritans,the theront cells also have the triplets constructed of the circumoral dikinetids and adjacent oral palps.The triplets correspond to the cytopharyngeal ridge,but overnumber the somatic kineties outside the cytostome.Toxicysts are absent in trophonts and protomonts and located in the oral palps and cytoplasm in oral region of theronts.Toxicysts may be involved in the life activity process of theronts infecting and invading into the host fish.Toxicysts are produced and differentiated in the daught cells in anaphase of the cell division inside tomonts.Trophonts,protomonts and theronts have the membranous folds,and there are somatic kineties between these folds.These folds may be caused by the close proximity of the somatic kineties in this area and high cytoplasmatic elevations covered by pellicle between them.They do not connect directly with the cytostome and may be related to parasitic characteristics of C.irritans.Trophonts,protomonts and theronts all have the adoral brosses,which are three fragments of dikinetids and bear clavate cilia.The adoral brosse primordium can be observed in the tomites surface in anaphase of the cell division inside tomonts.Adoral brosse of C.irritans is unique and different from any other families of class Prostomatea.2 Organelles and strutures of pellicle and under pellicle,and cell nucleus of C.irritans of different life history stagesPellicular alveoli of C.irritans are complicated and different from the similar structure of free living ciliates,and with various morphology features in different life history stages.In the feeding period of trophont stage,the pellicular alveoli contain many materials,and some vesicles with double membrane can connect to outside through small holes formed by membrane fusion.Pellicular alveoli might be able to take liquid or small granular nutrition outside the cell into them,and the nutrition will be accumulated in them temporarily.Many filamentous materials will be produced in the pellicular alveoli and discharged out the cell in the trophonts that are leaving host.Some parts of the pellicular alveoli with the inclusion are broke away into the cell cytoplasm by stages before and after encystment.Pellicular alveoli are compressed with little inclusion during cell division inside the tomonts.Pellicular alveoli of theronts become swollen again,and the inclusion might come from the activity of Gogi apparatus and secretory vesicles.Mucocysts are absent in trophonts and protomonts and located under pellicle of theronts.They may be involved in the infecting and parasiting process of theronts into the host fish.Mucocysts are produced and differentiated in the daught cells in anaphase of the cell division inside tomonts.C.irritans have multiple contractile vacuoles.The ultrastructure of contractile vacuoles is various in different life history stages,and they might have different functions.The function in trophonts might be important.Macronucleus of C.irritans present different morphology features in different stages.The macronucleus fuse and change from moniliform to spherical during encystment,and the chromatin become dispersive.3 Tomont of C.irritansMultiple layers of C.irritans tomont present similar morphology feature and might be of similar composition.There is no secretion of encystation-specific secretory vesicles or extrusomes during formation of the cyst wall.The somatic cilia and the cytostome are present during encystment.During cell division inside the tomont,dividing daughter cells formed temporary cell chains with growing cilia and without oral primordia.New oral primordia are found in tomites in anaphase of the cell division.Cellular life activities in the C.irritans tomont are quite active,with large amounts of synthesis and differentiation of materials and structures,such as Golgi apparatus,toxicysts,mucocysts.4 The phylogenetic position of C.irritansThe membranous folds,adoral brosse and nematodesmata of C.irritans are different from other families and genus of prostmes.And C.irritans has special parasitic lifestyle and complicated life history,with molecular phylogenetic analysis evidance,this study supports the establishment of the family Cryptocaryonidae.And there are even many specific cell mophology features which are different from the order Prorodontida C.irritans now belong to.The molecular phylogenetic analysis supports that there is intraspecific variation in C.irritans.In conclusion,mophology features and possible function of cytostome and cytopharynx,toxicyst,membranous folds,adoral brosse,pellicular alveolus,mucocyst and contractile vacuole of C.irritans in each life history stages are revealed deeply and systematically.Some life acivities,especially those related to the parasitic characteristics of ciliates,are preliminarily clarified.Tomont phenomenon of C.irritans is described and discussed for the first time.The phylogenetic position of C.irritans is made clear and definition of morphology description of C.irritans is listed out.This study provides new basic data of cell morphology and basic biology of C.irritans.