Morphological And Ultrastructural Comparison For The Different Ribotypes Of Margalefidinium Polykrikoides And An Investigation On The Factors Affecting Encystment And Excystment

Margalefidinium polykrikoides is an important toxic and harmful algal blooms?HABs?-forming dinoflagellate.M.polykrikoides was initially described in1961 from Puerto Rico,Caribbean Sea,and was subsequently reported from many coastal waters around the world.Prior to 1990s,the presence of M.polykrikoides and its blooms were reported from several countries and regions only,such as South Korea,Japan,Hong Kong,and a limited area of the east coast of North America.In the past three decades,however,this species appeared to have expanded its geographic distribution greatly around the world and it has been reported from many new regions,such as the coastal waters of the mainland of China,the Philippines,India,Indonesia,Singapore,and the Arabian Gulf countries,the United States,Mexico,and Spain.In China,many small-scale blooms of M.polykrikoides were reported from the coastal waters of Hong Kong,Guangdong,Zhejiang,Jiangsu,Shandong,and Tianjin during 1984-2016.M.polykrikoides exhibited acute toxicity to fish,shellfish,and zooplankton.Blooms of M.polykrikoides have caused catastrophic mortalities of fish and other animals,and thus led to tremendous economic losses in aquaculture and fisheries and serious effects on marine ecosystems.Therefore,M.polykrikoides blooms have become an important marine ecological disaster and this species is regarded as one of the most important and most intensively investigated HAB species in the world.Previous studies have shown that there are different geographical populations of M.polykrikoides around the world,which are in fact genetically different in the large subunit?LSU?rRNA gene,and thus they were divided into 4 different ribotypes on the basis of difference in LSU rDNA sequences,namely,?1?East Asian ribotype,?2?Philippine ribotype,?3?American/Malaysian ribotype,and?4?Mediterranean ribotype.Although the differences in the LSU rRNA sequences among different ribotypes are high enough to separate them into different species,it is not yet unclear whether these ribotype populations have discernible and stable morphological features.Therefore,in this study,we compared the cell morphology and ultrastructure among three strains of M.polykrikoides?the Chinese strain MPJZBC3,the USA strain CP1,and the Malaysia strain MPCoKK23?isolated from Jiaozhou Bay,China,the Paconic Bay of New York,the USA,and Malaysia.We confirmed at first that the strain MPJZBC3 from Jiaozhou Bay belongs to the East Asian ribotype,while both the American and Malaysian strains belong to the American/Malaysian ribotype?the identity between the Chinese strain and the USA/Malaysian strains is 93.6%,i.e.,1426 bp/1523 bp?.Importantly,however,in all commonly-used diagnostic characteristics in morphology and ultrastructure?cell size,single cell or colony and number of cells in a colony,shapes of epicone and hypocone,cingular displacement?numbers of turns encircling the cell?,shape of sulcus,shape of apical groove,position and shape of nuclei,shape and number of chromosomes,shape,structure,number and distribution of chloroplasts,and shape of pyrenoid,etc.?,there was no discernible difference among the three strains,i.e.between the East Asian ribotype and American/Malaysia ribotype.Therefore,we believe that,in order to obtain identification traits of different ribotypes,further intensive comparative studies should be conducted among all ribotypes of the species in terms of their life histories,morphologies of resting cysts,and stable physiological characteristics such as toxin species and toxicity potency,and sexual compatibility.At present,in the field monitoring of harmful algal blooms,particularly the monitoring for alien species,a reliable identification of M.polykrikoides should be based on the LSU rDNA sequencing.Numerous studies have shown that resting cyst plays an important role in the initiation and decline of algal blooms via germination and formation of cysts respectively.Recent studies have found that three ribotypes of M.polykrikoides can produce resting cyst,but the regulatory factors for cyst formation and germination have not been systematically investigated yet.In this study,four factors that have been shown to be important regulating factors for the formation of resting cyst or resting stage cells in other species of dinoflagellates and microalgae were investigated for the possible regulating effects on the cyst formation in M.polykrikoides,including nitrogen limitation,initial cell density,presence of predators,and calcium(Ca²⁺)concentration.Our results showed that:1)Nitrogen-limited cultures?1N/100-1N?promotes the formation of cysts,initial cell density,the presence of predators?Brachionus plicatilis?and calcium ions can affect the formation of cysts.2)Adequate nutritional conditions can promote cysts germination,the highest rate of cyst germination at 25?.Our results summarized above provide important understandings about the regulating roles of the ambient factors affecting cyst formation and germination of M.polykrikoides,which may provide further insights into the bloom dynamics of M.polykrikoides.The results also provide a scientific basis for subsequent research on the prediction,prevention and control of algal blooms based on cyst distribution and abundance.