The Antioxidative Defense And Immune Responses Of Flounder (Paralichthys Olivaceus) Larvae And Juveniles To Seawater Acidification And Cadmium Exposure

Ocean acidification(OA)and marine pollution are two globally marine environmental problems that have increasingly come to the foreground and public notice in past years.Ocean acidification not only directly affects the reproduction,development,growth and survival of marine organisms,but also influences the biotoxicity of pollutants,such as heavy metals.Therefore,the issues how OA and heavy metal exposure affect marine organisms at early life stages(ELSs)have become hot research topics in marine ecotoxicology studies.In this paper,a series of laboratory experiments were conducted to investigate how physiological life processes(e.g.,hatching,development,growth,survival,antioxidative defense,immune function and biomineralization)of the flounder Paralichthys olivaceus at ELSs(embryos,larvae and juveniles)responded to OA and cadmium(Cd)exposure.Subsequently,the integrated biomarker response(IBR)was established and used to evaluate the intergrative effects of the two stressors on the physiological processes of the flounder larvae and juveniles.This study could help improve the assessment of the toxic effects of the two stressors on the recruitments and population dynamics of fishery species.Additionally,the results are expected to provide knowledge for better understanding how marine fishes at ELSs respond to OA scenarios in the future.(1)Seawater acidification(pH 7.70 and 7.30)and Cd exposure(0.01,0.15 and0.50 mg L^-1 Cd²⁺)could inhibit the hatchability and yolk absorption and increase the morphological abnormalities of the larvae,leading to decreases in larval growth and survival.Cd exposure would reduce the activity of embryos and induce the protease hydrolysis,resulting in the abnormal functions of hatching-related enzymes and thus decreasing hatching success.Seawater acidification could affect the above-mentioned physiological and biochemical processes induced by Cd exposure.This could further cause a delay in hatching or aggravate the embryonic mortality,leading to low hatchability.The morphological deformity of the larvae mainly occurred in the skeleton(e.g.,spine and tail).The deformity might be related to the down-regulated oc(osteocalcin)gene and affected CA(carbonic anhydrase)and Ca-ATPase activities under seawater acidification and Cd exposure,which would hinder the absorption of calcium(Ca)and lead to a decrease of myosin and sarcomere and aggravate the developmental malformation in the skeleton of the larvae.(2)The Cd accumulation in the larvae or in the tissues(liver,gill and muscle)of the juveniles was postively dose-depndent on Cd and tissue-specific.The Cd accumulation increased following the order of liver>gill>muscle.However,seawater acidification did not affect the Cd accumulation either in the larvae or in the tissues of the juveniles.The liver is the main organ for detoxification and metabolism of the metals.The Cd that was absorbed by skin,gill and intestine was eventually transported into the liver and kidney for detoxification through the blood and lymph circulations.The Cd²⁺could bind to the-SH in the reduced glutathione(GSH)and metallothionein(MT),and is synthesized to low toxic compounds.The compounds could remain in the tissues for a long time and increase the Cd accumulation in the livers.The gill is the main organ for respiration,osmotic regulation and acid-base balance regulation of the juveniles.Seawater was continuously filtered to provide sufficient dissolved oxygen or maintained the osmotic balance and acid-base balance,increasing the Cd accumulation in the gills.The Cd in the muscles mainly comes from metal transfer via physiological metabolic processes such as blood and lymph circulations,which is commonly at relatively low concentration.Moreover,muslces have mass dilution effects due to their relatively large mass,potentially reducing the Cd accumulation level in muscles.Meanwhile,the tissue-specific Cd accumulation could affect the antioxidative and immune responses of the larvae and juveniles to the interactive stresses of the two stressors.(3)The antioxidative defense,immune responses and biomineralization of the flounder larvae were significantly affected by seawater acidification(pH 7.70 and7.30)and Cd exposure(0.01 and 0.15 mg L^-1 Cd²⁺).Specifically,increasing Cd²⁺concentration significantly induced the reduced glutathione(GSH)contents,the activities of superoxide dismutase(SOD)and glutathione S-transferase(GST),but inhibited the activities of catalase(CAT)and glutathione peroxidase(GPx).On the other hand,the antioxidants responded differently to seawater acidification,depending on biomarker or Cd²⁺concentration.Seawater acidification would affect the responses of antioxidative defense to the Cd exposure.Additionally,both seawater acidification and Cd exposure induced lipid peroxidation(LPO)and aggravated the oxidative damages to the flounder larvae.When it comes to immune responses,lysozyme(LZM)activities were reduced and immunoglobulin M(Ig M)contents were induced under seawater acidification and Cd exposure.These findings indicated that the two stressors resulted in an immunosuppression in the innate immunity and an immunostimulation in the acquired immunity in the larvae.Heat shock protein 70(HSP70)contents were induced by Cd exposure and seawater acidification,whereas MT contents were induced by Cd exposure but were not affected by seawater acidification.As for biomineralization and acid-base regulation,the acitivties of the three investigated enzymes that are related to biomineralization(CA,Na/K-ATPase and Ca-ATPase)in the larvae were all significantly induced under seawater acidification and Cd exposure.(4)The antioxidative defense and immune responses in the liver,gill and muscle of the flounder juveniles were all significantly affected by Cd exposure(0.01,0.30and 3.0 mg L^-1 Cd²⁺),which were apparently tissue-specific.Liver commonly responded more sensitively to the intergrative stress of the two stressors than gill and muscle,potentially being related to the tissue-specific Cd accumulation.However,seawater acidification did not show significant effects on the antixodative and immune responses in the liver,gill and muscle of juveniles.Specifically,in the liver,all the investigated antioxidants(SOD,CAT,GSH,GST,GPx)and immune biomarkers(LZM,ACP,AKP and Ig M)were inhibited at the highest Cd²⁺concentration(3.0 mg L^-1).In the gill,the activities of CAT,GST,GPx,LZM and ACP and GSH contents were all inhibited,but the activities of SOD and AKP and Ig M contents were induced at the highest Cd²⁺concentration.In the muscle,the activities of SOD,GST,GPx,ACP and AKP and GSH contents were all induced at the highest Cd²⁺concentration,but either the activities of CAT and LZM or the Ig M contents were not affected by Cd exposure.Additionally,both MT content and LPO level were significantly increased at the highest Cd²⁺concentration in all tissues.The inhibition of the antioxidative or immune biomarkers in the liver and gill might result from the high Cd accumulation in the two tissues,which could destroy the protein structures,inhibit the emzyme activities and consequently affect the physiological and biochemical processes.The Cd accumulation in the muscle was low compared to that in the liver and did not exceed the level at which the antioxidative and immune systems could cope with.Under such circumstances,the activities or contents of the antioxidative and immune biomarkers were induced so that the cells could be protected from oxidative damages.Overall,seawater acidification significantly affected the activities of CAT in the liver and gill and ACP in the muscle,but did not show significant effects on the other antioxidative or immune responses in any tissue of the juveniles.This could be possibly related to the fact that juveniles had developed relatively perfect mechanism of acid-base regulations.The impacts of excessive CO₂ and H⁺in acidified seawater on the juveniles could be timely defended by the functioning of CA,buffer ions and transmembrane proteins of the fish.Nonetheless,seawater acidification increased the antioxidative and immune responses of the juveniles to Cd exposure.(5)IBR index was established and used to assess how antioxidative defense or immune system and biomineralization of the flounder larvae and juveniles responded to the intergrative stress of seawater acidification and Cd exposure.The related biomarkers of the larvae and juveniles obviously responded more sensitively to the intergrative stress than that by a single stressor.In other words,seawater acidification could increase the toxic effects of Cd exposure on the flounder larvae and juveniles.Furthermore,the larvae responded more sensitively to the intergrative stress than the juveniles.IBR could more reliably reflect the comprehensive biotoxic effects of muti-stressors on tested organisms than a single-biomarker assessment.