Molecular Ecological Study Of The Response Of Heterosigma Akashiwo To Different Environmental Conditions

In the marine ecosystem,phytoplankton not only serve as the base of the food web,but also play an important role in biogeochemical cycles.However,some of species form exceptional blooms and cause significant damages to local economies,marine ecosystems and human health.These events are known as harmful algal blooms(HABs),and are commonly called Red tides because some blooms can discolor the water in red or brown.The raphidophyte Heterosigma akashiwo is a globally distributed harmful alga that has been associated with fish kills in coastal waters.Previous studies have demonstrated that many environmental factors can potentially regulate this species bloom formations,but what molecular mechanisms or biochemical processes are involved remain unresolved.In this study,the reference genes for gene expression analysis in H.akashiwo were evaluated under different environmental conditions.Using 18S rDNA metabarcoding to follow the shift of the eukaryotic phytoplankton community structure over our study period and conducted comparative metatranscriptomic analysis to understand the shift of major metabolic processes.Several laboratory culture experiments were also carried out to understand the response of H.akashiwo to environmental change(e.g.light and nutrient).The results as follows:(1)A group of genes that have been used as reference genes in various algae were summarized,and ten candidate reference genes were selected.The statilities in expression of these reference genes in H.akashiwo were analyzed under four different exprimental confitions.GeNorm and NormFinder evaluated the expression stability of these genes.The results showed that calmodulin(cal),P-tubulin(tub),60S ribosomal protein rpL17-2 and rpL23 are suitable reference genes for gene expression analysis under all the four different experimental conditions we examined.For further validation,these selected genes were used to normalize the expression levels of ribulose-1 5-bisphosphate carboxylase/oxygenase large unite(HrbcL)over a diel cycle.The validated reference genes will be useful for future gene expression studies on H.akashiwo,we expect that the procedure used in this study may be helpful to future efforts to screen reference genes for other algae.(2)To analyze phytoplankton community structure,the V4 variable regions of 18S rDNA gene were selected.The results showed that the phytoplankton community shifted from high diversity in the pre-bloom stage to H.akashiwo predominance during the bloom.The other stramenopiles(all stramenopiles except H.akashiwo)remained in low relative abundances,the growth of diatom might have been negatively influenced by the high abundance of H.akashiwo.Interestingly,the relative abundance of total dinoflagellates also increased along with H.akashiwo.(3)Metatranscriptome sequencing was used to identify how microorganisms adjust their metabolic processes in response to bloom formation.A sharp decrease in ambient dissolved inorganic phosphate and strong up-regulation of phosphate and dissolved organic phosphorus(DOP)uptake genes in H.akashiwo from pre-bloom to bloom.The observed up-regulated expression of mixotrophy related genes suggests potential contribution of mixotrophy to the bloom.Accelerating photosynthetic carbon fixation was also implied by the up-regulation of carbonic anhydrase genes during the bloom.In S.costatum,many chloroplastic ribosomal proteins,ATP synthase and photosynthesis genes were down-regulated during the bloom formation.Notably,we also observed a strong morning-to-afternoon shift in the expression of many genes involved in several pathways,such as photosynthesis,carbon fixation,DNA replication and citrate cycle.This result indicates that timing of sample collection should be taken into consideration for a field study aimed at comparing expression levels of genes over a multi-day period.(4)Understanding H.akashiwo gene expression patterns under different environmental conditions.Different blue light receptor Aureochrome(Haaureo)genes showed different expression patterns in light:dark(LD)cycle,only Haaureo1 exhibited a clear diel rhythm.When the cultures were transferred from LD cycles to continuous dark,the diel rhythm of Haaureol mRNA disappeared,indicating that Haaureol transcription is probably driven by dark-to-light transitional cues and in turn regulates expression of other genes in the light dark cycle.Under phosphorus and nitrogen limited condition,the expression levels of these nutrients uptake genes were up-regulated,indicating H.akashiw can utilize different nutrient sources.The transporters of ammonium and urea were up-regulated in dark period and this will benefit H.akashiwo as it can uptake more nutrient at lower depths.The results in this study also suggest that H.akashiwo cell division is probably regulated by the light:dark cycle through the diel regulation.