Effects Of Microcystis Aeruginosa Stress On Growth And Development Of Daphnia Similoides Sinensis And Screening Of Molting Related Genes

Since the 1980s,the eutrophication of Lake Chaohu in Anhui province has been at higher levels,and Microcystis blooms occur frequently.The interaction between cladocerans and Microcystis has become a research hotspot.Daphnia similoides sinensis is a dominant species in cladocerans in Lake Chaohu.Molting is usually an important physiological phenomenon in the life cycle of crustaceans(such as cladocerans).Some molting genes in crustaceans have been screened out,but the molecular mechanism on the molting of Daphnia species is still unclear.Other studies have shown that M.aeruginosa can significantly inhibit the growth and development of Daphnia species,and then interfere with their molting.In this study,D.similoides sinensis from Lake Chaohu was as research object,and the mixture of Scenedesmus obliquus and M.aeruginosa was as the food,and transcriptome sequences of D.similoides sinensis in three instars before/after the first pregnancy(namely,one instar before the 1^thadult instar,1^thadult instar and 2^thadult instar)were studied and the candidate genes related to molting were screened.The purpose of this study is to reveal the molecular mechanism on D.similoides sinensis molting,and to provide a reference for further investigation on gene function related to molting in Daphnia species.The results are as follows:1.With the increase of M.aeruginosa concentrations,the growth time of D.similoides sinensis in three instars before/after the first pregnancy gradually shortened.The presence of M.aeruginosa shortened significantly the time at first pregnancy and the time at first reproduction of D.similoides sinensis(P<0.001).The growth time at one instar before the 1^thadult instar was shorter than those at the 1^thadult instar and2^thadult instar.The first pregnancy of D.similoides sinensis in the M0 treatment(control)appeared at the 6^thinstar,whereas it all occurred at the 5^thinstar in the treatments containing M.aeruginosa(M2 and M4).With the increase of M.aeruginosa concentration,the body length of D.similoides sinensis in three instars before/after the first pregnancy became significantly shorter(P<0.05),and the eggs number at first pregnancy reduced significantly(P<0.05).Moreover,some individuals with abnormal molting were observed in the treatments containing M.aeruginosa.2.During the experiment,D.similoides sinensis in three instars(SP:one instar before the 1^thadult instar,SM:one instar at the 1^thadult instar,SA:one instar at the2^thadult instar)in the M0 treatment,and those in three instars(SP:one instar before the 1^thadult instar,SM:one instar at the 1^thadult instar,SA:one instar at the 2^thadult instar)in the M2 treatment were selected for transcriptome sequencing.18978unigenes were obtained,and the length of N50 was 4432bp with an average length of2138bp.In the non-redundant(Nr)protein database,the highest percentage of matching bases(80.5%)in species was to D.magna.GO enrichment analysis showed that,in the differentially expressed genes(DEGs)of D.similoides sinensis,both up-regulated genes and down-regulated genes in MP vs SP were focused on structural constituent of cuticle,the up-regulated genes in MM vs SM were mainly concentrated on structural constituent of cuticle,and the down-regulated genes in MA vs SA were focused on chitin binding.Moreover,the up-regulated genes in SA vs SM and down-regulated genes in both SM vs SP and SA vs SP were all mainly enriched in structural constituent of cuticle.The up-regulated genes in MA vs MP were concentrated on structural constituent of cuticle and structural molecule activity,whereas the down-regulated genes in MA vs MP were enriched on structural constituent of cuticle,chitin binding and structural molecule activity.3.In the three food treatments,the relative expression levels of Cluster-540.2485,Cluster-540.900,Cluster-540.7298,Cluster-540.9829,Cluster-540.3396,Cluster-540.4653,Cluster-540.8037,Cluster-540.7063,Cluster-540.9291 and Cluster-540.3989 of D.similoides sinensis in one instar before the 1^thadult instar were higher than those in the other two instars(P<0.05).The relative expression levels of Cluster-540.9000 in one instar before the 1^thadult instar in the M0 and M2treatments were higher than that in the other two instars(P<0.05).The relative expression levels of Cluster-540.8242,Cluster-540.6498 and Cluster-540.6920 in one instar before the 1^thadult instar in the M0 and M4 treatments were higher than those in the other two instars(P<0.05).Compared with the M0 treatment,in the treatments containing M.aeruginosa(M2 and M4),the relative expression levels of Cluster-540.8242,Cluster-540.6498,Cluster-540.9291,Cluster-540.6920 and Cluster-540.3989 were significantly up-regulated in the three instars(P<0.05).According to experimental results and functional annotation of differentially expressed genes,fourteen genes,Cluster-540.2485,Cluster-540.900,Cluster-540.9000,Cluster-540.7298,Cluster-540.8242,Cluster-540.9829,Cluster-540.6498,Cluster-540.3396,Cluster-540.4653,Cluster-540.8037,Cluster-540.7063,Cluster-540.9291,Cluster-540.6920 and Cluster-540.3989 in D.similoides sinensis may be regarded as candidate genes related to molting.The known nine molting genes(Ec R,E75,HR38,FTZ-f1,HR3,USP,shade,nvd1 and nvd2)were also found in the DEGs of D.similoides sinensis transcriptome in our study,and the relative expression levels were obviously different under M.aeruginosa stress.Compared with the M0 treatment,the relative expression levels of Ec R,E75 and FTZ-f1 in the three instars before/after the first pregnancy were significantly down-regulated in the treatments containing M.aeruginosa(M2 and M4)(P<0.01).The relative expression levels of HR38 and USP were significantly down-regulated in both one instar before the 1^thadult instar and at the 2^thadult instar(P<0.001).The relative expression levels of HR3 and shade were significantly down-regulated only in one instar before the 1^thadult instar(P<0.01),whereas the relative expression levels of nvd1 were significantly up-regulated in both one instar at1^thadult instar and at the 2^thadult instar(P<0.01).The relative expression level of nvd2 was significantly up-regulated in one instar at 1^thadult instar(P<0.05),whereas it was down-regulated in one instar at 2^thadult instar(P<0.001).In general,the growth and development of D.similoides sinensis was significantly inhibited in the presence of M.aeruginosa.In this study,under the M.aeruginosa stress,candidate genes related to molting in D.similoides sinensis are screened,and it will enrich biological information in ecdysis of cladocerans.