| Sea cucumber Apostichopus japonicus belongs to Echinodermata,was mainly distributed in the coastal areas of northern China and the shallow seas of the western North Pacific.It is one of the important economic marine aquaculture species in China,and it is an increasingly important part of world trade.Temperature and dissolved oxygen are two of the most important abiotic limiting factors in aquaculture of sea cucumber.In recent years,many sea cucumbers have died due to extreme environments in coastal areas and ponds in China,causing severe losses of resource and economy.That limited the sustainable development of aquaculture of sea cucumbers.Moreover,in the context of global climate change and intensified human activities,the phenomenon of high temperature and low oxygen in oceans is showing a normalization trend.Therefore,it is urgent to understand the response mechanisms of marine organisms to environmental changes.In order to ascertain the effects of high temperature and low dissolved oxygen on the body's physiological behavior and molecular regulation in extreme environment,the present study used sea cucumber as a typical echinoderm model to study its response mechanism to adverse environment.The temperature controller and self-designed real-time control system of dissolved oxygen simulated the high-temperature and low-oxygen environment in the experiments,respectively.The sequencing technique and real-time quantitative polymerase chain reaction(real-time PCR or q PCR)were used to verify and validate the changes and effects of m RNAs,long non-coding RNAs(lnc RNAs),micro RNAs(mi RNAs),proteins,metabolites and key immune factors in sea cucumbers in response to thermal and hypoxic stress,as well as the physiological and behavioral response by measuring key enzyme activities and behavior observation.The main findings are as follows:1.Physiological and behavioral responses of sea cucumbers under thermal and hypoxic stressHere,the physiological and behavioral responses of A.japonicus to high temperature and oxygen deficiency was illustrated.Sixteen enzymes related with digestion,immune defense and oxidative response were measured.AMS,PEP,TRY and LPS were significantly depressed in sea cucumber under environmental stresses,thus leading to a negative impact on digestive function.Moreover,the immune defense mechanism was induced based on the increased ACP and LZM.Furthermore,the activities of antioxidant enzymes(CAT,SOD and PPO)were changed to scavenge harmful substance like O2-,H2O2 and OH-.The antioxidant system was formed to reduce the oxidative stress.Moreover,the distribution,move speed,excretion and feeding of sea cucumbers were changed under environmental stress.Sea cucumbers were mainly distributed near water surface,and the body became edematous and the body wall became ulcerated and dissolved.In addition,compared with single stress factor,the tolerance of A.japonicus decreased sharply under the combined stress of heat and hypoxia.A.japonicus regulated digestion,immunity,oxidative stress and other related enzymes,forming a series of adaptive mechanisms and changed physical behavior to survive and adapt to the extreme environment.2.Changed m RNAs(message RNAs)and lnc RNAs(long non-coding RNAs)in sea cucumbers in response to thermal and hypoxic stressHere,differentially expressed(DE)lnc RNAs and m RNAs were identified as responses to environmental stresses in the economically important sea cucumber,Apostichopus japonicus.Based on a total of 159,355 and 495 significantly upregulated genes and 230,518 and 647 significantly downregulated genes identified in the thermal(HT),hypoxic(LO)and combination thermal+hypoxic(HL)stress treatments,respectively,we constructed DE-lnc RNA-m RNA coexpression networks.Among the networks,eight shared pairs were identified from the three treatments,and based on the connectivity degree,MSTRG.27265,MSTRG.19729 and MSTRG.95524 were shown to be crucial lnc RNAs.Among all the significantly changed lnc RNAs identified by real-time PCR and sequencing data,binding sites were found in four other lnc RNAs(MSTRG.34610,MSTRG.10941,MSTRG.81281 and MSTRG.93731)with Aja-mi R-2013-3p,a key mi RNA that responds to hypoxia in sea cucumbers.The hypoxia-inducible factor(HIF-1?)was also shown as the possible targeted m RNA of Aja-mi R-2013-3p.As indicated by a dual-luciferase reporter assay system,“HIF-1?gene/Aja-mi R-2013-3p/MSTRG.34610”network and the“HIF-1?gene/Aja-mi R-2013-3p/MSTRG.10941”network may play important roles in sea cucumbers under environmental stresses.Moreover,environmental stress altered the expression of multiple lnc RNAs and m RNAs,thus affecting various biological processes in A.japonicus,including immunity,energy metabolism and the cell cycle.At the molecular level,more comprehensive responses were elicited by the combined thermal/hypoxic stress treatment than by individual stresses alone in sea cucumbers.3.Changed micro RNAs in sea cucumbers in response to thermal and hypoxic stressHere,based on the construction of mi RNA profiles in sea cucumber under varied environmental stress,the DE-mi RNA,as well as the biological processes they involved in,were identified and characterized.Compared with normal condition,a total of 21,26 and 22 differentially expressed(DE)mi RNAs were clarified in sea cucumber A.japonicus under thermal,hypoxic and the combined stresses.In all,54 non-repetitive mi RNAs were identified to be differentially expressed in the pairwise comparison among three treatments with significant levels.The comparative mi RNA-seq and real-time PCR were used to identify and validate the representative mi RNAs,including Aja-mi R-novel-299,Aja-let-7b-3p,Aja-mi R-71b-5p,Aja-mi R-novel-13218,Aja-mi R-2004in response to high temperature and Aja-mi R-92b-3p,Aja-mi R-210-5p,Aja-mi R-novel-26331 in response to oxygen limitation.The potential targeted genes of DE-mi RNAs mainly involved in biosynthesis,metabolism,immunity,apoptosis and signaling transduction,suggesting that these processes are potentially impact the status of A.japonicus.4.Changed proteins in sea cucumbers in response to thermal and hypoxic stressHere,based on isobaric tags for relative and absolute quantification(i TRAQ),comparative proteomic analysis was used to illustrated proteins responded to varied environmental stress in sea cucumber.A total of 8437 proteins were identified,262,155and 433 proteins were differentially regulated in response to heat,hypoxia,and heat plus hypoxia,respectively.More differentially regulated proteins were identified under two environmental stress factors than single factor,indicating that the interaction between these two stressors has an additive effect at the proteome level.Based on the Gene Ontology(GO)analysis,proteins related with immune and defense response were largely induced and protein synthesis ability was depressed.Proteins involved in energy metabolism were significantly changed,including amino acid,carbohydrate and lipid metabolic processes.Additionally,hypoxic stress induced proteins involved in iron homeostasis.Gluconeogenesis was induced under heat and the combined stress.Alterations of defense strategies in sea cucumber are revealed to be normally dependent on the type of stressor.These proteome-wide alterations,including“substance biosynthesis,transport and metabolism”,“signal transduction”,“protein synthesis”,“immune and defense response”and“energy production and conversion”reflect an altered strategy that may enable A.japonicus to survive under global climate change in the future.5.Changed metabolites in sea cucumbers in response to thermal and hypoxic stressHere,ultra-performance liquid chromatography-mass spectrometry(UPLC-MS)was utilized to obtain metabolic profiles of A.japonicus under environmental stresses.Changes in the concentrations of 84,68,and 417 metabolites related to the responses of sea cucumbers to heat,hypoxia and the combined stress,respectively,were observed and analyzed.Synergistic effect of environmental stressors has an additive impact on the primary metabolome in sea cucumbers.Ten representative potential biomarkers of heat and hypoxia in sea cucumber are illustrated,including deltaline,fusarin C,cyclopiazonic acid,acroptilin,ophiobolin A,halichondrin B,absinthin,rapanone,volicitin.Results showed that metabolic processes related to amino acids,carbohydrates,lipids,cofactors and vitamins,and nucleotides responded to the environmental stresses,allowing the sea cucumber to maintain basic subsistence.As the results showed that,heat stress leads to a decreased carbohydrate and metabolism and changes in metabolites involved in amino acid,lipid,cofactors and vitamins metabolism.Moreover,when exposed to hypoxia,metabolites involved in amino acid,carbohydrate metabolism and the levels of fatty acids were significantly increased in sea cucumbers.The concentration of metabolites involved in the regulation of energy metabolism and the tricarboxylic acid(TCA)-cycle was significantly altered.Based on the decreased accumulation of glutamine,we hypothesized that heat stress is the main factor that interferes with the process of glutamic acid-glutamine metabolism in sea cucumbers.These results would facilitate further research on the adaptation of marine organisms to adverse environment.6.Time course analysis of immunity-related gene expression in the sea cucumbers in response to thermal and hypoxic stressThe results of the previous parts showed that the immunity of A.japonicus responded to the environmental stresses.Here,time course analysis of immunity-related gene expression in the sea cucumber A.japonicus during exposure to thermal stress and hypoxic stress were carried out.The expression trends of 17 genes related to the nuclear factor?B(NF-?B)pathway,the protease family,the complement system,heat shock proteins(HSPs)and the transferrin family during exposure to two stresses(high temperature and low dissolved oxygen)at eight time points were concluded.These genes have interconnected roles in immunity of sea cucumber defensing against environmental stresses.The expression levels of genes relating to the NF-?B pathways and HSPs were strongly affected in the sea cucumber thermal stress response,while melanotransferrin(Mtf),ferritin(Ft)and mannan-binding C-type lectin(MBCL)were affected by hypoxia.In contrast,complement factor B(Bf),myosin V(Mys)and serine protease inhibitor(SPI)were not that sensitive during the initial period of environmental stress.Similar expression patterns under both thermal and hypoxic stress for certain genes,including an increase in Hsp90 and decreases in lysozyme(LZM),major yolk protein(MYP)and cathepsin C(CTLC)were observed in sea cucumbers.Conversely,NF-?B and Hsp70 were differentially affected by the two stress treatments.Lysozyme-induced immune defense was inconstant in sea cucumbers coping with stress.Our analysis suggests that sea cucumbers demonstrate complex and varied immune responses to different types of stresses.These results provide information about the innate immunity of A.japonicus and represent a key immune index of a marine organism under environmental stress.The findings in this study facilitate an understanding of the adaptive strategies of holothurians in coping with global climate changes. |
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