| It is a special biological phenomenon with highly scientific value that unicellular green algae live within the endodermal epitheliomuscular cells of Hydra sinensis.The symbiotic relationship between host and symbiotic algae and the symbiotic mechanisms such as the compatibility,interaction,metabolic flow and energy flow between them are the focus of the related research of this field.In our previous study,one aposymbiotic strain of H.sinensis was prepared after the symbiotic algae were "knocked out" from the symbiotic strain of H.sinensis by special methods.Interestingly,this strain without symbiotic algae can reproduce asexually and survive for a long time by feeding Artemia sp.larvae,and this phenomenon indicates that symbiotic algae are not necessary for the survival of H.sinensis.Because of this,we guess that symbiotic algae may transfer some of their photosynthetic products to host cells as a nutrient supplement for hydra under special circumstances such as starvation.To confirm this hypothesis on molecular biology level,we cultured symbiotic strain of H.sinensis under starvation stress in three different photoperiod conditions: 24L:0D(continuous light exposure),12L:12D(light exposure for 12 h,darkness for 12 h within a 24-hour cycle)and 0L:24D(continuous darkness).And then the comparative transcriptomic and metabolomic analysis were carried out between light exposure treatment groups(24L:0D group and 12L:12D group)and dark treatment group(0L:24D group).The main results are summarized as follows:1.Molecular responses of H.sinensis under starvation stress to different photoperiods based on transcriptomic analysis155.7 Gb Clean Data were obtained by transcriptome sequencing,meanwhile 601,077 transcripts were spliced and 322,881 unigenes were assembled.Based on unigene expression analysis,32,400 differentiallyexpressed genes(DEGs)were identified between 24L:0D group and 0L:24D group,including 8,969 up-regulated genes and 23,431 down-regulated genes.According to these DEGs,384 KEGG metabolism pathways were enriched.There were 479 DEGs(164 up-regulated and 315 down-regulated)involved in26 carbohydrate metabolism pathways,442 DEGs(143 up-regulated and 299down-regulated)involved in 32 lipid metabolism pathways and 380 DEGs(139up-regulated,241 down-regulated)involved in 22 nitrogenous compound metabolism pathways.Among these 384 metabolism pathways,the first 50 metabolism pathways ranked by enrichment significance included 10 carbohydrate metabolism pathways,5 lipid metabolism pathways,7nitrogenous compound metabolism pathways,2 antioxidant-related pathways and ABC transporter.In addition,28,825 DEGs were identified between 12L:12D group and0L:24D group,including 11,198 up-regulated genes and 17,627down-regulated genes.According to these DEGs,384 KEGG metabolism pathways were enriched.There were 461 DEGs(201 up-regulated,260down-regulated)involved in 26 carbohydrate metabolism pathways,433 DEGs(181 up-regulated,252 down-regulated)involved in 33 lipid metabolism pathways and 369 DEGs(182 up-regulated,187 down-regulated)involved in20 nitrogenous compound metabolism pathways.Among these 384 metabolism pathways,the top 50 metabolism pathways ranked by enrichment significance included 9 carbohydrate metabolism pathways,4 lipid metabolism pathways,8nitrogenous compound metabolism pathways,3 antioxidant-related pathways and ABC transporter.2.Molecular responses of H.sinensis under starvation stress to different photoperiods based on metabolomic analysisGas chromatography-mass spectrometry(GC-MS)was used to comparative metabolomic analysis of 24L:0D,12L:12D and 0L:24D group of H.sinensis,then 1,156 features were detected and 402 substances were identified.In addition,the multidimensional and unidimensional analysis were combined to screen the differential metabolites between groups,and the results showed that there were 197 differential metabolites between 24L:0D and0L:24D group,including 31 carbohydrates(26 up-regulated,5 down-regulated),52 lipids(29 up-regulated,23 down-regulated)and 42 nitrogenous compounds(15 up-regulated,27 down-regulated).Based on these 197 differential metabolites,60 metabolism pathways were enriched,and there were 24 differential metabolites(23 up-regulated,1 down-regulated)involved in 9carbohydrate metabolism pathways,33 differential metabolites(25up-regulated,8 down-regulated)involved in 15 lipid metabolism pathways and54 differential metabolites(20 up-regulated,34 down-regulated)involved in 18 nitrogenous compound metabolism pathways.In order of the enrichment significance,ABC transporter was 2nd of these 60 metabolism pathways,and the antioxidant-related glutathione metabolism pathway was the 3rd.Furthermore,166 differential metabolites were identified between12L:12D and 0L:24D group,including 26 carbohydrates(18 up-regulated,8down-regulated),36 lipids(21 up-regulated,15 down-regulated)and 41 nitrogenous compounds(15 up-regulated,26 down-regulated).Based on these166 differential metabolites,54 metabolism pathways were enriched,and there were 19 differential metabolites(15 up-regulated,4 down-regulated)involved in 9 carbohydrate metabolism pathways,27 differential metabolites(16up-regulated,11 down-regulated)involved 13 lipid metabolism pathways and52 differential metabolites(22 up-regulated,30 down-regulated)involved in 16 nitrogenous compound metabolism pathways.In order of the enrichment significance,ABC transporter was the 1st of these 54 metabolism pathways,and the antioxidant-related glutathione metabolism pathway was the 12 th.3.Biological significance of molecular response of H.sinensis to light exposure treatment(relative to continuous darkness treatment)under starvation stressThe results of transcriptomic and metabolomic analysis showed that H.sinensis under starvation stress had six molecular responses to light exposure treatment(relative to continuous darkness treatment):(1)Active carbohydrate metabolism was associated with symbiotic algae transmitting some of their photosynthate to hydra cells as nutritional supplements for their hosts;(2)Active lipid metabolism implied that lipids might also be involved in nutrient transport from symbiotic algae to hydra cells,but whether photosynthate(carbohydrates)is converted into lipids in symbiotic algae and then transferred to hydra cells or photosynthate is first transferred to hydra cells and then converted into lipids for nutritional storage remains to be studied;(3)Pentoseand glucuronate interconversions pathway was enriched based on differentially expressed genes and differential metabolites,which was the direct evidence for the mutual transformation of carbohydrates and lipids in H.sinensis;(4)Active nitrogenous compound metabolism may be associated with the conversion from nitrogenous wastes(normal cell must excrete nitrogenous wastes out of itself)retained within hydra cells to some nitrogenous compounds that were harmless to hydra and also could be transferred easily to symbiotic algae as nitrogen source for essential plant nutrients;(5)Active ABC transporter system confirmed that there was a certain scale of metabolic flow between host and symbiotic algae to maintain the symbiosis relationship in H.sinensis under starvation stress during light exposure treatment;(6)A large amount of reactive oxygen species(ROS)from the photosynthesis of symbiotic algae was difficult to diffuse from the closed symbiotic vacuole to external water environment,and ROS can also be transmitted to host cells,then over-accumulation of ROS can damage cells.Therefore,these antioxidant-related pathways enriched based on DEGs and differential metabolites may be involved in controlling and scavenging ROS. |
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