Combined Toxic Effects Of Seawater Acidification And Trace Metals(Cd²⁺?Cu²⁺) On Economic Shellfish

Since the industrial revolution,anthropogenic activities such as fossil fuel combustion and deforestation have released significant amounts of anthropogenic carbon dioxide(CO2)into the atmosphere.At the same time,the ocean,which accounts for 70%of the Earth's surface area,absorbs about 30%of the carbon dioxide exhaled by humans,causing lowering of seawater pH and disequilibrium of carbonate/bicarbonate buffer system.This process is termed ocean acidification(OA).In coastal waters,seawater acidification does not exist in isolation,but it often coexists with other environmental stressors such as trace metal pollution,seawater warming and hypoxia,which makes the environmental stresses faced by offshore ecosystems more complicated and diversified.Despite this,most of the studies focus on the action of a single stressor on organisms,while the combined effects of multiple stressors are less studied.Marine bivalves constitute a major taxonomic group in estuarine and coastal regions,and play important roles in ecosystem functioning and economic value.Thus,the purpose of this study was to elucidate the toxic mechanism of seawater acidification and trace metal exposure to bivalves,and to provide a scientific basis for environmental monitoring and ecological risk assessment of trace metal pollution under global climate change.The results were summarized as followed:(1)The accumulation and subcellular distribution of cadmium(Cd2+)and the physiological responses including antioxidative response,histopathological changes and apoptosis of the oyster Crassostrea gigas were investigated after 31 days of exposure to OA and/or Cd2+,either alone or in combination.Increased Cd2+accumulation was found both in gills(about 57%increase in Cd2+ content at pH 7.8,22%increase in Cd2+ content at pH 7.6)and digestive glands(about 38%increase in Cd2+ content at pH 7.8,22%increase in Cd2+ content at pH 7.6)of C.gigas under seawater acidification.Meanwhile,a higher partition of Cd2+in the BIM(biologically inactive metal)fractions of gills(about 60%)was found in Cd2+-exposed treatments compared to the digestive glands(about 45%).The difference in Cd2+ subcellular distribution patterns between gills and digestive glands perhaps could explain their different sensitivity to Cd2+ and seawater acidification.Moreover,synergetic effects of Cd2+and OA on the oxidative stresses,histopathological damage,and apoptosis of exposed oysters were observed in this study(p<0.05)(2)In order to evaluate the defense responses of oysters under the combined exposure to OA and cadmium,the oysters C.gigas were exposed to Cd2+ and/or OA for 31 days.Results showed that OA exposure alone led to increased DNA damage,apoptosis rate and ROS production of hemocytes.However,inhibited phagocytosis rate,combined with increased DNA damage,apoptosis rate and ROS production of hemocytes were observed in oysters under exposure to Cd2+ exposure alone or combined with OA(p<0.05).Significant interactive effects between OA and Cd2+were observed on ROS production and DNA damage of hemocytes.In addition,there is generally significant increase in the mRNA expression of genes related to immune-related TLR pathway(Toll-1,I?B,Rel,MyD88 and TRAF3)and two immune factors(TNF and integrin beta-1B)in Cd2+-exposed oysters at pH 7.6,and synergetic effects of Cd2+and OA on most of the immune-related genes(Toll-1,MyD88,TRAF3,SOD,HSP90 and TNF)were observed in this study(p<0.05).The results revealed that depressed hemocyte function under OA and Cd2+ perhaps sensitized oysters to potential pathogen infection.(3)Physiological parameters,histopathological damage and condition index,oxidative stress and neurotoxicity biomarkers,combined with glycolytic enzymes activities were investigated in C.gigas exposed to Cu2+ and/or OA for 14 and 28 days.The bioconcentration of Cu2+ was increased in soft tissues of Cu2+-exposed oysters under OA.Our results suggest that both OA and Cu2+ could lead to physiological disturbance,oxidative stress,cellular damage,energy metabolism disturbance and neurotoxicity on oysters.The inhibited CR,increased glycolytic enzymes activities and decreased CI suggested the energy metabolism strategy adopted by oysters was not sustainable in the long term.Damages including vacuolization,hypertrophy,cilia erosion and intracytoplasmic inclusions of eosinophilic granules occurred in oyster gills in response to Cu2+ and/or OA,and OA severely increased the damage index(pH 8.1+Cu2+=0.9;pH 7.8 +Cu2+=1.0;pH 7.6 + Cu2+=1.4)of Cu-exposed oysters.Furthermore,integrated biomarker response(IBR)results found that OA and Cu2+exposure lead to severe stress to oysters,and co-exposure was the most stressful condition.(4)To investigate whether the toxicity of Cu2+ increases due to elevated Cu2+bioaccumulation under acidified seawater indiscriminately in two bivalve species from different coastal habitats.This study evaluated the effects of exposure to Cu2+and/or OA on two bivalve species(Manila clam Ruditapes philippinarum and Zhikong scallop Chlamys farreri)for 28 days.The contents of Cu2+ in soft tissues of clams and scallops increased under acidified seawater.Our results clearly showed that Cu2+ toxicity increased under acidified seawater by affecting the molecular pathways,physiological function,biochemical responses and health status of both clams and scallops.An iTRAQ-based quantitative proteomic analysis revealed increased protein turnover rate,disturbed cytoskeleton and signal transduction pathway,increased apoptosis and suppressed energy metabolism pathway in both clams and scallops under jointly exposure to OA and Cu2+.IBR results suggested that scallops were more sensitive to the toxicity of Cu2+ and/or OA than clams.Proteomic results suggested that increased energy metabolism and suppressed protein turnover rates might contribute to higher resistance to OA in clams than scallops.