Transgenerational Exposure To Ocean Acidification Increases Mercury Toxicity In Pseudodiaptomus Annandalei

Over the past 250 years,the atmospheric concentration of carbon dioxide(CO2)has been increased by nearly 40%(i.e.,from the preindustrial level of 280 ?atm to the present concentration with 400 ?atm),which was mainly caused by human activities especially including fossil fuel burning.As an important sink of CO2,ocean can absorb approximately one third of this greenhouse gas emission in the atmosphere,hence giving rise to increased seawater acidity and lower pH value,that is,ocean acidification(OA).As projected,the seawater pH value will continue to decline to around 7.6-7.9 by the end of this century(2100).Ocean plays an important role in the global mercury cycle.After a huge mass of mercury(Hg)is released by human activities,these pollutants will finally be end up in the oceanic areas mainly via dry/wet precipitation and land-surfac runoff,resulting in marine Hg pollution.Please note that Hg is a global pollutant with great toxicity,persistence and high mobility.In the current day,mercury pollution mostly occurs in coastal areas,but with the continuous emission from human activities,marine mercury pollution will become increasingly serious and will threaten more and more marine organisms.As a consequence,in ocean,when marine biota are exposed to OA for a long time,the transgenerational effect of OA may modulate the response of the progeny to Hg pollution.In terms of transgenerational effect,it means the experience of the parental generation can shape the phenotype of their offspring.Marine copepods greatly contribute to marine plankton and thus play a vital role in marine ecosystem.Also they are considered as a good testing organism for marine stress ecology because of their small size,short generation and strong fertility.Among them,Pseudodiaptomus annandalei is one of the few marine copepods that can be maintained for many generations in the laboratory,which has been a good model species in marine stress ecology.In this study,marine copepod P.annandalei was conditioned to the present(400?atm,Control)and near-future(1000 ?atm,OA)pCO2 levels for three consecutive generations(F0-F2),followed by one-generation exposure to different OA and Hg pollution in a crossed-factor manner(i.e.,Control:pCO2 400 ?atm+Control(C-C),pCO2 1000 ?atm+Control(C-O),pCO2 400 ?atm+1.0 pg/L Hg(C-H),and pCO2 1000?atm+1.0?g/L Hg(C-OH);OA:pCO2 400 ?atm+Control(O-C),pCO2 1000?atm+Control(O-O),pCO2 400 ?atm+1.0 ?g/L Hg(O-H),and pCO2 1000 ?atm+1.0?g/L Hg(O-OH)).Three important life-history traits(development time to maturation,number of nauplii/clutch,and total fecundity)were examined for F0-F2 and mercury accumulation analysis for F3.Specifically,quantitative proteomics was performed for the copepods of F3 to examine critical functional proteins and their biological processes,and via comparisons among the proteomes from different groups(C-C,C-O,C-H,COH,O-C,O-O,O-H,and O-OH),we sought to understand how the copepod P.annandalei responded to OA and Hg pollution at both molecular and phenotypic levels after transgenerational exposure to OA.The main findings are described as follows:(1)Long-term multigenerational exposure to OA displayed the negative effect on reproduction of marine copepod P.annadalei,and furthermore,the adverse impact tended to increase with generations.However,after one-generation recovery in clean seawater(O-C),the reproductive capacity of P.was significantly increased compared with the ambient group(C-C);thus,transgenerational exposue to OA enabled the copepod to display strong phenotypic plasticity.The above statement was further evidenced by proteomic analysis for O-C vs C-C.That is to say,due to transgenerational effect of OA,P.annadalei decreased many macromolecule synthesis and metabolic process so as to save more energy,which can be compensatory response to resist "longterm OA stress",a hypothetical information primed from their parents.The increased energy was in turn used for reproduction in the copepods,and the consequence is that the reproductive capability was significantly improved under O-C vs C-C.(2)After transgenerational exposure(F0-F2)to the near-future pCO2 condition,OA stress(i.e.,O-O vs C-O)significantly prolonged the development time of P.annadalei,while Hg pollution(O-H vs C-H)not only retarded its development but also restrained the reproduction of P.annadalei.Given transgenerational exposure to OA,P.annadalei down-regulated numerous biological processes to cope with the "long-term OA" transmitted by the parents,and thus was in a vulnerable state;as a result,its resistance to additional stress was likely reduced.In this context,after transgenerational exposure,OA stress(O-O vs C-O)significantly decreased pyruvate metabolism,cell growth,peroxisome and such-like processes,consequently inhibiting growth and reproduction of P.annadalei.Similarly,Hg pollution(i.e.,O-H vs C-H)significantly interfered with many important biological processes in P.annadalei,such as TCA cycle,cytochrome P450,ribosomes and cell protein metabolism,ultimately resulting in significantly negative effects on its development and reproduction.(3)Under both the present and near-future pCO2 conditions,when compared with Hg exposure,combined exposure of OA plus Hg significantly reduced Hg accumulation and its toxic effect in the copepod P.annadalei.In other words,OA alleviated Hg toxicity to the development and reproduction of P.annadalei.Under combined exposure of OA and Hg,more stress response and detoxification processes including the enhanced lysosome and phagosome,increased organic metabolism,and upregulation of TCA cycle and glycolysis/gluconeogenesis were initiated in P.annadalei to counteract Hg toxicity;that is,despite transgnerational exposure(F0-F2)to different pCO2 conditions,growth and reproduction of the F3 copepods were significantly increased under combined exposure when compared with single Hg treatment.(4)When the transgenerational exposure to OA(increasing Hg toxicity)and OA effect(decreasing Hg toxicity)were simultaneously acting(i.e.,O-OH vs C-OH),the net effect was the reduced Hg toxicity to the development and reproduction of P.annadalei.The possible reason for OA reducing Hg toxicity may mainly be a chemical effect as exemplified by cation competition between proton and Hg at binding sites and decreased membrane permeability due to low pH.Conversely,transgenerational effect means the informantion passed from the parents to their offspring,thus more being a biological effect.In our study,when these two effects were co-occurring,the chemical effect seemed stronger than the biological effect;that is to say,the OA effect was more significant.Therefore,the Hg accumulation,development time,number of nauplii/clutch,and total fecundity were significantly increased in the C-OH vs O-OH case.In summary,this work reveals how marine copepods,in the mechanistic review,respond to environmental stresses(such as OA and Hg pollution)after transgenerational exposure to OA,and it should facilitate more accurate prediction for the impact of global changes and metal pollution on marine ecosystem in the future ocean.