Physiological,Biochemical And Molecular Responses Of Copepods With Diverse Ecological Traits To Diatoms

Diatoms are important primary producers in marine ecosystems,while copepods are the main components of primary consumers.Zooplankton transfer substance and energy from the bottom of the food chain to the advanced predators by feeding on phytoplankton.Therefore,the classic food chain theory holds that "diatom-copepodfish" is the principal pathway of substance circulation and energy flow in marine ecosystem.However,studies in recent decades have verified that certain diatoms have negative impact on larval development,egg production and egg hatching of copepods,and this phenomenon is common both in field and laboratory,which made the classic food chain theory to be debatable.Since copepods utilize diatoms more efficiently will increase copepod population,while the latter has a significant impact on fishery resources.Therefore,it is of great importance to study the effects of diatom on the survival and reproduction of copepods.Three copepod species with diverse ecological traits including:Acartia pacifica,Pseudodiaptomus annandalei and Tigriopus japonicus were selected in present study,differences in survival rate,reproduction and antioxidant enzyme activity of copepods were analysed,and the differences in gene expression patterns of copepods responded to diatoms were also compared to investigate whether different copepods respond to diatoms are species-specific.In addition,in order to explore the possible adaptation mechanism of copepod to diatoms,P.annandalei were cultured with two diatoms for consecutively four generations and the indicators above were determined.The main research results are as follows:1)Survival rate of copepods with diverse ecological traits were remarkblely influenced by diatom species and concentrations(P<0.05).When Acartia pacifica nauplii fed on Chaetoceros mulleri,nauplii successfully developed to adult,though survival rate is obviously inhibited at high concentration of diet(P<0.05),while the nauplii could not develop to adult when fed on Nitzschia closterium f.minutissima.The survival rate of nauplii droped sharply since the fourth day of experiment,and the downtrend would be more drastic with diatom concentration increasing.Both C.mulleri and N.closterium f.minutissima supported the development of nauplii to adults in P.annandalei,however,survival rates were significantly lower than control(P<0.05).The survival rate of P.annandalei was in direct proportion to C.mulleri concentration while inversely proportional to N.closterium f.minutissima concentrations.Compared to the control,C.mulleri showed a negative effect on egg production and egg hatching of A.pacifica and significantly correlated with diatom concentrations:when C.mulleri was offered at a concentration of 0.35 ?gC·ml-1,egg production rate and hatching success were 10.7±4.8 Eggs·female-l·d-1 and 66%respectively,which were similar to the control(P>0.05);when diatom concentration increased to 1.70?gC·ml-1,egg production rate decreased to 3.1±3.4 Eggs·female-1·d-1 and no eggs hatched.Reproduction of P.annandalei fed on N.closterium f.minutissima were significantly lower than that on C.mulleri and the control(P<0.05).When Pseudodiaptomus annandalei was cultivated for 4 generations on the two diatoms at 1.7 ?gC·ml-1,It was shown that starting at the second generation,P.annandalei exhibited adaption to Chaetoceros muelleri and N.closterium f.minutissima with improved survival.2)Antioxidant enzyme activities including GPx,SOD and GST of three species of copepods feeding on two diatoms were determined,the results showed that the GPx activity of A.pacifica was not influenced by C.mulleri while SOD and GST was significantly induced,so are GST induced by N.closterium f.minutissima(P<0.05);As to P.annandalei,both SOD and GST activities were induced by two diatoms,while only higher concentration of diatoms resulted in higher GPx activity;Antioxidant enzyme activities in T.japonicus were comparable to control when fed on C.mulleri and lower concentration of N.closterium f.minutissima,while SOD activity was increased to 167.1± 14.8 and 163.4±7.9 U·mgPr-1 at high Nitzschia closterium f.minutissima concentration(8.50 and 17.00 ?gC·ml-1)respectively.No significant changes of GPx activity in P.annandalei was observed among four generations,so are SOD and GST activities of P.annandalei when C.mulleri was offered as diet;However,when P.annandalei fed on N.closterium f.minutissima,both SOD and GST activities showed a trend of decreasing with the progress of generations,indicating that SOD and GST may play an important role for P.annandalei in the process of adaptation to diatoms.3)Fluorescence quantitative PCR(RT-qPCR)technique was used to explore the changes of MnSOD,GST and HSP70 gene expression level in copepods fed on different microalgae in present study.Species specific of gene expression paterns were observed on three species of copepods fed on the same diet:MnSOD and GST expression level of A.pacifica increased at lower diatom concentration but decreased at higher concentration.C.mulleri showed no effect on MnSOD and HSP70 expression level of P.annandalei,and N.closterium f.minutissima showed significant inhibitory effect.Microalgal species rather than concentration were correlated to gene expression of T.japonicus,gene expression level were not affected by C.mulleri,while that of MnSOD and HSP70 were significantly induced.Different response patterns of three species of copepods fed on diatoms may lead to different survival rates and reproduction among copepods.P.annandalei may adapted to N.closterium f.minutissima with improved gene expression of MnSOD and HSP70 when P.annandalei was cultured for generations on N.closterium f.minutissima.