Exploring The Molecular Mechanism Of Wing Dimorphism In The House Cricket (Acheta Domesticus)

Wing dimorphism is a common phenomenon in insect morphology polymorphism.It's widely existed in insect orders such as Diptera,Heteroptera,Coleoptera and Orthoptera.Wing dimorphism mainly divided into“inherent wing dimorphism form”and“wing-shed wing dimorphism form”.Wing dimorphism is an adaptative strategy for insects to environmental changes,and this morphological plasticity has rised many entomologists'interests.Exploring on the mechanism of wing dimorphism could provide a scientific explanation for the understanding of adaptive evolution of insects.However,previous studies focus on only a few species of insects such as planthoppers and aphids,moreover,there are relatively few studies on molecular basis of the wing dimorphism of Orthoptera especially the insects with“wing-shed wing dimorhism form”.Acheta domesticus Linnaeus 1758 is in the family Cricketidae of Orthoptera.The wing dimorphism in A.domesticus is formed by wing loss.It is easy to distinguish the winged and wing-shed ones,thus the wing dimorphism of A.domesticus is an ideal model to study the“wing-shed wing dimorphism form”.In this study,transcriptome analysis,WGCNA,RNA interference and other experiments were used to explore the molecular basis of wing dimorphism in A.domesticus.The results were listed as follows:1.Wing dimorphism in A.domesticus can be classified as“wing-shed wingdimorphism form”.The hind wings of the crickets fully developed and beyond the abdomen during the early stage after emergence.Some crickets began to shed their hind-wings after eclosion,the wing-shed peak in A.domesticus occurred at the 20~thh day after eclosion,and the wing-shed ratio start to decrease after day 36,the dynamic of wing-shed in male and female crickets were similar.Additionally,there is no visible phenotypic differences could be observed between winged and wing-shed house crickets.2.The results of transcriptome analysis for eight stages of A.domesticus.RNA libraries were prepared based on the eight developmental stages:1~stt instar nymphs(stage I),2~ndd instar nymphs(stage II),3~rdd instar nymphs(stage III),new emerged adults(stage IV),10-day-old adults(stage V),20-day-old wing-shed adults(stage VI),30-day-old adults(stage VII)and 40-day-old adults(stage VIII).We obtained approximately 135.23 Gb of clean reads from the RNA libraries.All samples had quality scores at the Q30 level higher than91.35%.A total of 141,456 Unigenes were obtained after de novo assembly.Of these unigenes,18,771 were exceeded 1,000 kb in length.Through annotation with NR?Swiss-Prot?KEGG?COG?KOG?GO and Pfam database,32,508 unigenes were matched to known proteins.3.DEGs analysis on different insect stages of A.domesticus.We divided insectstages into three groups for the comparison of DEGs:comparison A was between before wing-shed peak(IV+V)and wing-shed peak(VI),comparison B was between wing-shed peak(VI)and after wing-shed peak(VII+VIII),while comparison C was between after(VII+VIII)and before(IV+V)wing-shed peak.Volcano plot,MA-plot and Heatmap suggested an increasing number of DEGs at the wing-shed peak(stage VI).The DEGs were further enriched and clustered by GO,COG and KEGG analyses to examine the biological processes/pathways.The results showed that many DEGs were enriched in“Amino acid transport and metabolism”,“ribosome,purine metabolism”and“protein processing in endoplasmic reticulum”,suggesting these DEGs are mainly involved in protein synthesis and interaction during the growth and development of the house crickets.4.Weighted correlation network analysis(WGCNA)analysis showed some genes may regulate wing dimorphism in A.domesticus.This analysis grouped the 21,922 DEGs into 18 modules based on their similarities in expression trends across the eight developmental stages,and labeled the 18 modules with different colors.The red module was the most positively relevant module to the wing-shed occurrence profile among the 18modules.In the 15 profiles of the red module,the expression profile 9 was selected as the key profile since it's the closest to the wing-shed occurrence profile and contained the most DEGs.23 DEGs were screened for the gene co-expression network using Cytoscape with 0.45 value.The DEGs AdomCFDP(craniofacial development protein),AdomDIS3L(DIS3-like exonuclease)and AdomHSP40(heat shock protein)were shown to have same expression profile as the occurrence of wing shedding with the top three MCODE scores.In addition,as the FPKM of Nylanderia fulva virus was much higher than orthers genes of the profle 9,this gene was also selected as a candidate gene.We supposed that the wing-shed in the house crickets is due to infection by N.fulva virus.So,there were total four candidate genes that may directly regulate the wing dimorphism of A.domesticus were screened out by WGCNA.5.RNAi analysis of candidate genes and phenotypic analysis:(1)Insulin receptors are not involved in the wing-shed of the house crickets.Previous studies have shown that insulin receptors can regulate the wing dimorphism of planthoppers.The insulin receptor(AdomInR:Insulin receptor)was identified from the transcriptome data of housed crickets.And the RNAi result revealed that the wing shedding was not affected by the insulin receptor,suggesting that the molecular mechanisms of wing dimorphism in house cricket is differ from those in N.lugens and reflect a different determination patterns of wing dimorphism between two species.(2)HSP40 mediates wing shedding in the house crickets.The five candidate genes(AdomCFDP?AdomDIS3-L?AdomHSP40?AdomInR and Nylanderia fulva virus)were succesfully interfered with dsRNA-injection.Phenotypic analysis of the five dsRNA-injected insects revealed that the wing-shed ratio of adult house crickets was significantly influenced only by dsRNA of AdomHSP40 compared with those of the wild-type and dsGFP-injected groups.The expression of AdomHSP40 was up-regulated during the wing-shed peak period of the house cricket,which promoted wing shedding,While when the expression of AdomHSP40was reduced by RNAi,the wing-shed rate of the house crickets decreased.This result indicated that AdomHSP40 was involved in regulating the wing dimorphism by wing-shed.The result of Fluorescence in situ hybridization(FISH)assay showed the AdomHSP40 was expressed at the base part of hindwings,the expression level of AdomHSP40 was higher in the wing-shed peak period than that of stage IV and sage VIII.The FISH results further demonstrated that HSP40 play a crucial role in the wing dimorphism by wing-shed of the house crickets6.The comparisons of the song and genitaliabewteen the winged andwing-shed house crickets.There was significant difference of the duration of single pulse and the intervals between winged and wing-shed crickets,which indicated that the hind-wings shedding had some influence on the calling songs by the fore-wing of crickets;However,there were no significant changes in the morphology of testis and ovary between the winged and wing-shed crickets.This study use WGCNA approach of and find that AdomHSP40 played a crucial in the wing shedding of the house crickets.Which provide us some insights into the molecular mechanisms underlying wing dimorphism in the house crickets A.domesticus.