| Mayflies are unique and valuable aquatic insects,with a long series of plesiomorphies and automorphies.Among these characters,the prometabolan developing pattern(four stages in life history,egg,nymph and two winged instars,subimago and imago)is remarkable.However,the origin of prometabola and functions of subimagos are unsolved and molecular studies remain to be performed.The mayflies in the genus Cloeon(Ephemeroptera:Baetidae)are good candidates for those researches because:(1)They distribute widely in ponds,pools or other lentic waters around the world so it is easy to collect;(2)The morphological changes of them are obvious and they are good representatives of the order;(3)The nymphs of Cloeon can live very long in the lab,so they can be reared and observed conveniently.In this study,we choose the species Cloeon viridulum to explore the peculiarity of prometabola and the significance of subimagos.In the present research,we want to not only clarify the taxonomy of the species Cloeon viridulum morphologically,but also get some knowledge of its ecology.Further,we try to discuss the phylogeny of Ephemeroptera in the Pterygota and relationships between prometabola and other types of metamorphosis upon mRNA and mitochondrial DNA sequences.Our study includes the following four sections:1.Morphology and taxonomy of Cloeon viridulumThe species Cloeon viridulum was named by Navas in 1931 based on the female subimago(holotype)collected in Shanghai.However,the original description is quite short and simple,with only a very low-quality drawing picture.Since then no any other report has been made.Here,upon numerous of Cloeon specimens collected from Shanghai,Jiangsu,Zhejiang and Anhui provinces and two female paratypes in the Museo de Zoologia del Ayuntamiento,Barcelona,Spain,we describe morphological details of all stages of C.viridulum and confirm its species status,which provide the base for following molecular studies.2.Life history of Cloeon viridulum and its related temperatureThrough the field collection,observation and indoor rearing,we observe the life history of C.viridulum,especially the emerging and molting process and time.Meanwhile,temperature of the pool water(the Caiyue Lake in Nanjing Normal University)and population dynamics are recorded.In the lab experiments,the individuals of C.viridulum rearing at different temperatures(12?,20?,25?,30?)are counted every 12h(12h,24h,36h,48h,60h,72h)and the expression changes of heat shock protein(HSP40,HSP60,HSP70 and HSP90)are measured.The results show the highest survival ratio of C.viridulum is at 15?,which was close to the natural condition(lake temperature 18?).The expression changing of heat shock proteins is lowest at this temperature accordingly.3.The transcriptional signatures of Cloeon viridulum at different stagesThis study generates the first de novo transcriptome assembly of mayfly.The transcriptome of C.viridulum,based on 88 Gb of sequence data,containing a set of 81,185 high quality transcripts.The gene expression signatures among the young larva(YL),mature larva(ML),subimago(SI),and imago(IM)stages of this mayfly are compared.The number of differentially expressed genes(DEGs)in YL vs.ML,ML vs.SI,and SI vs.IM,is 4,825,1,584,and 1,278,respectively,according to the reads per kilobase of transcript per million mapped reads analysis,assuming a false discovery rate<0.05 and a fold change>2.Gene enrichment analysis reveals that these DEGs are enriched in the "chitin metabolic process","germ cell development","steroid hormone biosynthesis",and "cutin,suberine,and wax biosynthesis" pathways.The expression pattern of selected group of candidate signature genes for Prometabola,including vestigial,methoprene-tolerant,wingless,and broad-complex were confirmed by quantitative real time-PCR analysis.The Q-PCR analysis of larval,subimaginal,and imaginal stages of C.viridulum suggests that the development of mayflies more closely resembles hemimetamorphosis rather than holometamorphosis.4.Mitochondrial genome of C.viridulum and phylogeny of EphemeropteraWe use transcriptome data assembly and the specific primer amplification methods to sequence the whole mitochondrial genome of C.viridulum.The nearly complete mitogenome should be a typical circular DNA molecule by comparing with other mayfly mtgenomes.Totally 29 genes(12 PCGs,16 tRNAs,1 rRNAs)are sequenced and analyzed.In addition,mtgenomes of 3 mayflies(sequenced)and other 48 arthropods(download from GenBank,including 9 mayflies and 2 decapodans as outgroup)are adopted to reconstruct the phylogeny of major clades of Pterygota by using Bayesian inference(BI)and Maximum Likelihood(ML)methods.Both topologies show the Ephemeroptera is a monophyletic group although its position is not stable.Considering the condition of Hemiptera(members grouped into distant clades)and the advantages of Bayesian method,we incline to the BI tree.It shows that the Ephemeroptera diverges from others basally and developmental hypothesis is"epigenetic+(prometabola+(incomplete metamorphosis+complete metamorphosis))",which means the prometabola is more similar to hemimetabola but has its own specific characteristics.This result is consistent to that of the transcriptome analysis. |
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