Transcriptome Analysis And Screening Of Candidate Genes Related To Morphology Induced By Fish Pheromone In Daphnia Galeata

Cladocera is an important group in aquatic ecosystem,which plays a connecting role in the food chain.It can not only filter planktonic algae but also be predated by fish.Usually,higher fish predation pressure can trigger"induced defense"in Daphnia species,resulting in changes in morphology,behavior or other life-history characteristics.Therefore,the Daphnia species is considered to be a model organism for the response of prey to predator pheromone in phenotypic plasticity.Some morphology-related genes have been screened out in crustaceans,but the molecular mechanism of defense morphogenesis(especially helmet and tail spine)in Daphnia species remains unclear.In this study,different body parts(helmet,trunk body and tail spine)of Daphnia galeata were obtained,and the transcriptome sequencing and RNA interference techniques were used to screen the candidate genes related to the morphology of D.galeata under the induction of fish pheromone.The results of this study will provide a theoretical basis for revealing the molecular mechanism of the morphological changes of Daphnia under fish induction.The main results are as follows:1.To carry out transcriptome analysis of D.galeata,the helmet(BH),tail spines(BT)and body(BR)in the control treatment and the helmet(PH),tail spines(PT)and body(PR)in the fish pheromone treatment were selected.After mixed assembly,25825 unigenes in D.galeata were obtained.The relative species with higher homologous sequences were Daphnia pulex(46.1%)and Daphnia magna(32.5%).GO enrichment showed that the up-regulated genes in PT vs BT mainly focused on cuticle structural components and molecular activity.Both up-regulated and down-regulated genes in PH vs BH mainly focused on peptidase activity and endopeptidase activity.In BH VS BR,the up-regulated genes mainly focused on peptidase activity whereas the down-regulated genes mainly focused on extracellular region and peptidase activity.In BH VS BT,both up-regulated and down-regulated genes were mainly concentrated in the membrane.Both up-regulated and down-regulated genes in PH vs PR mainly focused on peptidase activity and proteolysis.Both up-regulated and down-regulated genes in PH vs PT mainly focused on protein metabolism and catalytic activity.2.The top 30 up-regulated differential genes in PT vs BT,PH vs BH,BH vs BR,BH vs BT,PH vs PR and PH vs PT transcriptomes were selected for q PCR verification,and a total of 24 candidate genes that may be related to morphological defense of D.galeata were obtained.The expression levels of 4 genes in PH were significantly higher than that in BH,PR and BR(P<0.05),whereas those in PT were significantly higher than those in BT,PR and BR(P<0.05).These genes may be involved in the morphological changes(e.g.the helmet and tail spine)of D.galeata.Two trypsin genes(Cluster-2384.9683 and Cluster-2384.9311)are closely related to molting process.The expression levels of the other 4 genes in PT were significantly higher than those in BH,BT,BR,PH and PR(P<0.01),which these genes may be involved in the tail spine changes of D.galeata.One cuticle protein gene(Cluster-2384.10979)may be closely related to the morphological defense of D.galeata.The expression levels of the remaining 16 genes in PH were significantly higher than those in BH,BT,BR,PT and PR(P<0.05),among which seven genes(e.g.Cluster-2384.10346,Cluster-2384.4417,Cluster-2384.7946,Cluster-2384.8263,Cluster-2384.9831,Cluster-2384.3878 and Cluster-2384.16699)may be involved in the helmet changes of D.galeata,which are associated with immunity and defense.In addition,two known genes that may be involved in morphological changes were also found in the D.galeata transcriptome(Cluster-2384.8096:acidic mammalian chitinase,Cluster-2384.8883:putative transcriptional factor distal-less protein).q PCR results showed that the expression of Cluster-2384.8096 and Cluster-2384.8883 in PH and PT were higher than in BH and BT,respectively(P<0.05),suggesting that the two genes were involved in the morphological changes of both helmet and tail spine in D.galeata.3.The expression of gene Cluster-2384.10979(Dagcut)related to cuticle in PT was significantly higher than those in BH,BT,BR,PH and PR(P<0.05).After ds RNA feeding interference experiment,know-down of Dagcut gene in D.galeata was obvious.Compared with the 1%ds GFP(E.coli without the gene of interest)and2%ds GFP treatments,the expression of Dagcut gene in both 1%dscut(E.coli containing the gene of interest)and 2%dscut treatments dropped significantly(P<0.05).In the RNAi experiment,37.5%of the 5^thto 7^thinstars of D.galeata mothers in the 1%and 2%dscut treatments died because of molting abnormality.In the 2%dscut treatment,the tail spines of D.galeata during embryogenesis even curved,and this phenomenon appeared also in living and dead newborns at second reproduction.In conclusion,the morphological change of D.galeata was observed in the presence of fish pheromone.Transcriptome analysis was used to screen the candidate genes related to the morphology of D.galeata,and the function of Cluster 2384.10979(Dagcut)gene was identified by RNAi technology.This study supply a basis for the exploration of the genes related to the morphological defense of cladocerans,and also provides a theoretical basis for revealing the molecular mechanism of the morphological change of cladocerans.