The Application Of Tyramide Signal Amplification-Fluorescent In Situ Hybridization In The Study Of Specific Nanoflagellate Groups

Nano flagellate(nanoflagellate,NF)is an important component of marine pico-and nanoplankton.As one of the key links of the microbial loop,it can not only regulate the abundance and community structure of picoplankton by grazing,but also contribute to the regenerative productivity of marine ecosystem by nutrient regeneration,which is of great significance in oligotrophic ocean area.However,due to the limitation of research approaches,scientists used to study nanoflagellate as a "black box",which leads to ignoring some of the key groups,especially,the dominant groups of nanoflagellate.Therefore,studying the spatio-temporal distribution and the grazing status of specific nano flagellate groups have crucial significance for people to recognize the internal world of nanoflagellate,explore the community composition of pico?and nanoeukaryotes and understand the role played by target groups in the microbial loop.Tyramide signal amplification-fluorescent in situ hybridization(TSA-FISH)is the only known method to quantify target groups at the cellular level.In this study,Micromonas and MAST-4 were used as the main research objects.We re-evaluate the specificity oligonucleotide probes for two specific groups of nano flagellate and explore the optimal hybridi-zation conditions.Subsequently,by using this method combined with the spring and summer voyages in the Taiwan Strait,we have study the spatial and temporal distribution and the grazing status of target groups,as well as their role in the composition of picoeukaryotic community.The main results are as follows:(1)Under the condition of three central mismatches,Micro 01 and NS4 can match 97.28%and 99.28%target sequences respectively,with relat:ively high specificity.Micro 01 had the best hybridization effect for Micromornas in 40%and 45%formamide concentration,while the optimal formamide concentration for probe NS4 was 35%and 40%.(2)Under the optimum hybridization condition,the quantitative analysis of Micromonas and MAST-4 in the surface seawater of the Taiwan Strait in spring was carried out by TSA-FISH and qPCR.The results showed that the number of rDNA copies per cell of Micromonas and MAST-4 were 8.5(R=0.201)and 9.4(R=0.587),respectively.However,due to the fewer samples,the linear correlation between cell abundance and copy number of rDNA was not obvious.(3)The horizontal distribution of target groups in the Taiwan Strait and northeastern South China Sea shows that the abundance of Micromonas decreased with the increase of offshore distance in surface water.In the DCM layer,the abundance of our target group in the northeastern South China Sea was significantly higher than that in the Taiwan Strait.In the bottom layer,the abundance of target group in the northeastern South China Sea decreased with the increase of offshore distance.In vertical direction,Micromonas had obvious stratification in profile B,which was shown as surface layer>DCM layer>bottom layer.In profile A and C,with the increase of offshore distance,the stratification became more and more obvious.At the coastal profile,the abundance of Micromonas decreased with the increase of water depth.The diurnal and nighttime vertical distribution of Micromonas at M3 station showed that,with the increase of water depth,the cell abundance increased first and then decreased,showing obvious vertical movement of this group in M3.(4)The results of the investigation on the composition of picoeukaryotic community at M3 showed that Micromonas is not only the dominant group in the Photosynthetic picoeukaryotes(PPE),but also the dominant organism in the whole picoeukaryotic community.The dominance of Micromonas was particularly evident in the euphotic zone.(5)The ingestion rates of Micromonas and MAST-4 to bacteria were 0.07 cells ind'1 h-1 and 1.36 cells ind-1 h-1,respectively,which was significantly lower than the mean ingestion rate of nanoflagellate(4.17 cells ind-1 h-1).It revealed that the ingestion rates of specific nanoflagellate groups were not only affected by the cell size,but also closely related to their nutritional types and illumination conditions.These results indicated that using of TSA-FISH technology to study specific nano flagellate groups can not only fill in the blanks of Micromonas and MAST-4 in the Taiwan Strait,but also help us to recognize the internal world of nanoflagellate and the role of specific groups in the composition of pico-and nanoeukaryotic communities.