Study On The Effect Of RHA-based Silicon Source Replenishment On Planktonic

Diatoms are dominant groups in coastal phytoplankton community structure and major contributors to Marine primary productivity,it is also an important natural food for filter-feeding shellfish during their growth and development.For diatom phytoplankton,silicate is an essential nutrient element during their growth and development.However,the phenomenon of seasonal silicate restriction in many typical aquaculture areas in China has adverse effects on the growth of diatom phytoplankton and also affects the growth of filter-feeding shellfish fed on diatom.China is a major country in mariculture in the world,and the output of shellfish aquaculture accounts for more than half of the total output of mariculture.In shellfish aquaculture,the highest output is filter-feeding shellfish such as oysters and clams.In this context,it is necessary to explore the ecological and safe methods to deal with silicate limitation.This paper selected a typical large-scale aquaculture bay of shellfish,Sanggou Bay,as a research area and took the dominant group of phytoplankton,diatoms,as research objects.Based on technical methods combined with physiological ecology and molecular biology,multiple experimental methods such as laboratory simulation and field isolation were used.Explore response of diatom community structure to liquid silicate enrichment in Sanggou Bay in spring,laboratory simulation study on the response of Nitzschia closterium to different concentrations of liquid silicate,study on the effect of RHA on diatom instead of liquid silicate,discussion on the slow release of silicates from RHA to natural sea area,selective feeding of four species of micro-algae by Philippine clams and their mechanisms.The results of this study can provide basic data and valuable clues for exploring the feasibility of silicon element replenishment strategies in the critical period of filter-feeding shellfish large-scale culture area,developing phytoplankton primary productivity enhancement techniques to improve shellfish culture capacity,and revealing the selective feeding mechanism of filter-feeding shellfish such as Philippine clams.1.Response of diatom community structure to liquid silicate enrichment in Sanggou Bay in spring.The response of the diatom community structure to silicate enrichment in Sanggou Bay in spring was studied by using ground-based enclosure experiments.The results showed that adding silicate in spring significantly increased the cell abundance of phytoplankton in Sanggou Bay.On the 7th day,chlorophyll a concentration in the LS group（low concentration silicate group）and HS group（high concentration silicate group）was increased by 95.00%and 94.00%,respectively,compared with the control group.The cell abundance of microphytoplankton and nanophytoplankton was significantly increased.In 2021,44 species of phytoplankton were identified in the in-situ seawater of Sanggou Bay,including 34 species of diatom,9 species of dinophyta and 1 species of chlorophyta.Diatoms,which account for 77.27%of the species richness of phytoplankton community structure in the in situ seawater of Sanggou Bay,are the dominant species,followed by dinophyta.The addition of silicate increased the abundance of diatom cells,and the abundance of diatom cells in the LS group and HS group was 1.55 times and 1.71 times of that in the Control group,respectively.Moreover,it can significantly improve the dominant species of phytoplankton in Sanggou Bay,such as nitzschia closterium,cyclotella sp.,and thalassiosira rotula.The addition of silicate significantly improved the phytoplankton community structure richness index and had no significant effect on the uniformity and diversity index.2.Laboratory simulation study on the response of nitzschia closterium to different concentrations of liquid silicate.The effects of different concentrations of silicates on the growth of nitzschia closterium under the condition of silicon enrichment were studied.The results showed that the higher the silicate concentration was,the higher the cell abundance was.The highest abundance was 1.35×10⁷cells/mL,which appeared in the HS group on the 10th day.The algal cell abundance in groups HS（high concentration silicate group）,MS（medium concentration silicate group）and LS（low concentration silicate group）was1.71,1.42 and 1.18 times of that in Control group,respectively.Silicate concentration can significantly affect the change of algal cell growth rate,the higher the silicate concentration,the longer the growth rate maintained positive,to the end of the experiment,the algal cell growth rate in groups HS and MS was positive,and cell abundance still maintained positive growth,LS group and Control group algal cell growth rate has been negative;When the concentration of silicate was 180μmol/L,the growth rate of nitzschia closterium remained positive during the experiment,and the growth rate remained at a higher level than that in other treatment groups and the maintenance time was longer than that in other treatment groups.3.Study on the effect of RHA on diatom instead of liquid silicate.The effects of adding RHA on the community structure and particle size structure of diatoms in Sanggou Bay in spring were studied by field ground-based containment experiments.The results showed that the addition of RHA could significantly increase the abundance of phytoplankton cells in Sanggou Bay in spring.Compared with the Control group on the 7th day,the chlorophyll concentration in the LR group（an addition amount of RHA:0.10 g/L）and HR group（an addition amount of RHA:1.00 g/L）increased by 82.54%and 96.03%,respectively.At the end of the 7th day,chlorophyll concentration in the LR and HR groups was 3.39 times and 7.61 times of that in the Control group,respectively.The addition of RHA increased the abundance of diatom cells,and the abundance of diatom cells in LR group and HR group was 1.44 times and1.67 times of that in the Control group,respectively,and it can significantly improve the dominant species of phytoplankton in Sanggou Bay in spring,such as nitzschia closterium,cyclotella sp.and Coscinodiscus spp..Adding RHA significantly increased the species richness index of phytoplankton community structure,but had no significant effect on the diversity and evenness index.4.Discussion on the slow release of silicates from RHA to the natural sea area.The slow release of silicates from RHA to natural sea areas was studied by laboratory ecological experiments.The results showed that when RHA was added into slow-release bag made of corn fiber,the period of silicate release from RHA could reach32 weeks,the average release rate from week 0-24 was 5.44μmol/（d·g RHA）,each gram of RHA releases 640.38μmol SiO₃^2-.The phosphate release period was 32 weeks and the average release rate during 0-18 weeks was 0.13μmol/（d·g RHA）,and 23.78μmol PO₄^3-was released per gram of RHA.When RHA is added to concrete instead of cement,a certain amount of silica-phosphorus can be released,and the release cycle of silicate and phosphate is 10 weeks.With 1%RHA,the average increase is 24.03μmol SiO₃^2-and 0.30μmol PO₄^3-.5.Selective feeding of four species of algae by Philippine clams and their mechanisms.The relative expression level of Rp-CTL gene in different tissues and organs of Philippine clams was determined by fluorescence quantitative method and the effect of starvation stress on Rp-CTL gene expression was determined by ion chromatography.Carbohydrate extraction and monosaccharide composition in B-EPS（adhesion extracellular polymers）of four microalgae such as Rp-CTL were determined by ion chromatography.The results showed that when four different microalgae were mixed together with the same density,the feeding rate of Philippine clam to different microalgae was different.The feeding rate of the Philippine clam was higher for nitzschia closterium and cyclotella sp.,while the feeding rate of chaetoceros curvisetus and prorocentrum minimum was lower.Rp-CTL gene was highly expressed in the feeding organs（gill,labial valve）and surface mucus of Philippine clam（P<0.05）,but hardly expressed in blood cells,seashells and other tissues and organs.Starvation stress could significantly increase the relative expression level of Rp-CTL gene in feeding organs.The increase of Rp-CTL gene expression under starvation stress may be the result of the Philippine clam to improve the capture and sorting efficiency of food particles.In this study,the glucose content in B-EPS sugar components of nitzschia closterium and cyclotella sp.with a high feeding rate was significantly higher than that of chaetoceros curvisetus and prorocentrum minimum with a low feeding rate.Therefore,it is inferred that the feeding selection mechanism of the novel C-type lectin in Philippine clam based on particle chemistry may be through the recognition of glucose in B-EPS on the cell surface of microalgae to mediate the preferential intake of food particles,but further research is needed to verify.