Evidence For Resting Cyst Production By Three Common Harmful Algal Blooms-forming Dinoflagellates And The Cyst Distribution In The Sediment Of China Coastal Waters

Dinoflagellates are a group of protists that cause about 75% of all harmful algal bloom(HAB)events over the world.Both environmental factors and the biological characters of the HABs-causing species contribute to the occurrence of HABs of dinoflagellates.Among all contributing biological characters,the life history of dinoflagellates,especially the resting cyst formation in many species,plays a vital role in the initiation and geographic expansion of HABs because the germination of resting cysts under favorable conditions can inoculate("seeding")an initial population in the pre-blooming seasons and the cysts can be more easily transferred,either via natural or anthropogenic(e.g.via ships' ballast water sediments)pathways,from one to another location.While about 200 among ca.2400 species of dinoflagellates have been documented to be resting cysts producers,the majority,including many HABs-forming species,have not been proved to be capable of cyst producing.Therefore,proving whether or not a dinoflagellate can form resting cysts is of particular importance in terms of providing a mechanistic explanation for the recurrence and geographic spread of HABs,and even in providing a basis for the forecasting the occurrence and scale of HABs based on mapping the distribution of resting cyst in marine sediments.Among all HABs-forming dinoflagellate,Karenia mikimotoi,Prorocentrum donghaiense,and Karlodinium veneficum are three of the most important ones as they often form density blooms in many regions of the world,including China,cause massive kills of many marine animals or destructive effects on the coastal ecosystems,and consequent economic losses.Except for some fragmental evidences,however,whether or not these three species can form resting cysts have not be systematically investigated yet.Hence,this dissertation studied the life histories of three species based on laboratory cultures and confirmed these three species capable of producing resting cysts both in laboratory cultures and the field.We also mapped the distribution of resting cyst in the marine sediment of China coastal waters using multiple approaches.The major findings of the work are summarized as follows:(1)Observations on the life histories of the three species based on laboratory cultures.We observed cell pairs mating,planozygotes,and resting cysts in all three dinoflagellates.The resting cyst of K.mikimotoi are characterized by a spherical shape,a smooth surface(without spiny process),a diameter ranging from 15.9 to 28.9 ?m,an obvious brown or pale brown accumulation body,and a thin cyst wall.The resting cyst of P.donghaiense are spherical or elliptic,with a smooth surface of no spiny process,a diameter ranging from 10 to 30 ?m,at least two layers of cyst wall,darker than the vegetative cells,and a brown accumulation body.K.veneficum cysts have a spherical shape,a smooth surface,a diameter ranging from 9.6 to 17.2 ?m,an orange or red accumulation body,a brown or light gray color,and a thick cyst wall(at least two layers).The cysts of all three species can be germinated in the laboratory,with a germination time requirement for K.mikimotoi cyst 3-6 days,about 3 days for P.donghaiense,and2-36 days for K.veneficum cyst,the first two have a low rate of germination(< 20%).(2)Establishment of a protocol for the detection of resting cyst in marine sediments and the detection results.In this thesis,a method combining PCR,fluorescence in situ hybridization(FISH),single cell isolation,and single-cell PCR and clone sequencing was used to detect resting cyst in marine sediment samples.First,specific primers of K.mikimotoi,P.donghaiense and K.veneficum were designed and tested against the 28 S r DNA D2 region,respectively.Second,the genomic DNA of the sediment samples collected from the locations where the blooms of the target species occurred was extracted,and PCR detections using the primers tested above were conducted to detect the presence of these three species in the sediments.Third,the respective FISH probes specifically designed against the 28 S r DNA D2 region for three species,with the 5' end of each probe labeled with either the fluorochrome FITC(fluorescing green)or Cy3(fluorescing orange)were applied to an optimized FISH protocol to detect the resting cysts from marine sediments and observed a epifluorescence light microscope.Finally,in order to further confirm the identity of cysts positively detected with both FISH probes labeled with FITC and Cy3,the fluorescing cysts were individually isolated and a single-cell PCR and subsequent sequencing was conducted.Based on the results of sequences,we confirmed that presence of resting cysts of K.mikimotoi,P.donghaiense and K.veneficum in the field marine sediments.The results fill up the vacancy of the life history and ecology in the three dinoflagellates,providing a reasonable explanation for the interannual recurrence and global geographical spread of the three dinoflagellates,and an important foundation for the early warning and prediction of the three dinoflagellates.(3)Establishment of a protocol for mapping the resting cysts in marine sediments along the coast of China sea.Together,125 sediment samples were selected for the detection,covering all four seas of China(Bohai Sea(BS),Yellow Sea(YS),East China Sea(ECS)and South China Sea(SCS))and with 8,22,70,and 25 sediment samples from BS,YS,ECS,and SCS,respectively.Real-time PCR(Taq Man)and FISH were both used to map the distribution of resting cysts in sediments of China coastal waters.Real-time PCR was used to quantify the abundance of cysts after taking into consideration the 2N ploidy of cysts and the measurements of r DNA copy number of three dinoflagellates both in vegetative cells and cysts.Also,because of the DNA loss during the process of DNA extraction,the q PCR results were adjusted based on the DNA extraction efficiency of vegetative cells(due to the low availability of cysts for the three species measuring the efficiency).One third of the q PCR products amplified from all sediment samples were further cloned and sequenced to confirm the specificity of assays.FISH was conducted to enumerate the resting cysts in 40 sediment samples also covering all four seas of China and to compare with the results obtained via q PCR.For K.mikimotoi,among the 125 sediment samples collected from all four seas of China,58 sediment samples(46.4 %)were detected positive for the presence of K.mikimotoi cyst.The calculated abundance of K.mikimotoi cyst ranged from 0 to 33cysts/32 g wet sediment.For FISH detection,the abundance of K.mikimotoi cysts varied from 0 to 14 cysts/32 g wet sediment across all 40 sample.Base on the results of the two methods,the abundance of K.mikimotoi cyst in ECS was the highest among all four seas,while no significant difference was found between ECS and SCS(P >0.05).For the numbers of samples detected positive,ECS was 65.7%(N=70),SCS was44%(N=25),YS was 9%(N=22),and BS was 25%(N=8).Regression analysis showed a significant correlation(P < 0.05)between the cyst abundance quantified using q PCR and that using FISH.According to the slope of the regression line,however,the cyst abundance obtained from FISH detection was generally higher than that from q PCR detection.Nevertheless,both methods(FISH and q PCR)detected a wide distribution of the K.mikimotoi cysts in the sediments of China seas but generally with a low abundance.For P.donghaiense,36 of the 125 sediment samples from all four seas of China were detected positive in the presence of P.donghaiense cyst(28.8%).The calculated abundance of P.donghaiense cysts ranged from 0 to 62 cysts/32 g wet sediment.For FISH method,the abundance of P.donghaiense cysts varied from 0 to 8 cysts/32 g wet sediment in 40 samples.Comparatively,the abundance of P.donghaiense cysts in ECS was the highest among all four sea areas(P < 0.05).For the numbers of samples detected positive,the detection rate in the ECS was 42.8%(N=70),the detection rate in the SCS was 20%(N=25),the detection rate in the YS was 4.54%(N=22),and the detection rate in the BS was 0%(N=8).The highest detection rate was found in ECS(42.8%).Regression analysis showed a significant correlation(P < 0.05)between the cyst abundance quantified by q PCR and that quantified by FISH.According to the slope of the regression line,indicating that cyst abundance obtained from FISH detections tended to be lower than that from q PCR detection.In spite of this,both methods(FISH and q PCR)showed an extremely low abundance of P.donghaiense cyst,a wide distribution in sediments of ECS,SCS,YS,and no distribution in BS,indicating the distribution of P.donghaiense cyst in China seas has a latitude boundary.For K.veneficum,among the 125 sediment samples collected from all four seas of China,94 sediment samples(75.2 %)were detected to be positive in the presence of K.veneficum cyst.The calculated abundance of K.veneficum cyst ranged from 0 to 25cysts/32 g sediment(wet weight).For FISH method,the abundance of K.veneficum cysts varied from 0 to 15 cysts/32 g sediment(wet weight)across all 40 sampling sites.Base on the results of two methods,the abundance of K.veneficum cyst had no significant differences across all sites that were detected(P > 0.05),but significantly varied among different sampling regions.The highest abundance of cyst was observed in ECS via two methods.For the numbers of samples detected positive,the detection rate in the ECS was 76.05%(N=70),the detection rate in the SCS was 76%(N=25),the detection rate in the YS was 81.82%(N=22),and the detection rate in the BS was75%(N=8).There are no significant differences among detection rate of sea area.Regression analysis showed a significant correlation(P < 0.05)between the cyst abundance quantified by q PCR and that quantified by FISH.According to the slope of the regression line,FISH detections generated higher levels of cyst abundance compared to q PCR detections.After all,both methods(FISH and q PCR)detected very low abundance but a wide distribution of K.veneficum cyst in the sediment samples of China seas.Our results summarized above demonstrated that the three HABs-forming dinoflagellates can produce resting cysts in the field(i.e.not only in laboratory)and the resting cysts widely distribute along the coastal sediment of all four China seas.Although the abundance of these cysts is generally very low in the sediments of most areas in comparison to those species well-known in having abundant cysts,their germinations are still enough to inoculate initial vegetative cell populations for bloom.Therefore,our detection results provide essential knowledge for the blooming dynamics of these HABs-forming dinoflagellates.