FIGURE LEGENDS
FIGURE 1 Orthologous genes and phylogenetic tree of insects. (a) Venn diagram of shared orthologous genes in WWS and six other insect species. All the numbers refer to the numbers of genes. Numbers in overlapped circles are the numbers of common genes between or among different species. (b) Phylogenetic tree and estimated evolutionary path for WWS and other invertebrates. The species with dotted lines represent hemimetamorphosis located in the lower branches while species with solid lines represent holometabola located in the upper branches. The circles shaded blue, light blue and gray areas represent the boundaries for the Ordovicain-Permian, Permian-Triassic and Jurassic to the present, respectively. The arrows on different order represent the direction of insect evolution.
FIGURE 2 Genome evolution of E. pela . (a) Distribution of WWS genomic features. Track a: Circular representation of the linkage chromosomes of the WWS genome. Track b–d: GC content (100kb window), gene density (100kb window) and percentage of repeats (100kb window). Track e-g: Mean expression of annotated genes calculated as log (FPKM + 1) of the mean expression value in three repeat female samples in the first larvae stage. (b) Genome wide Hi-C interaction map with 500k resolution. The color from light to deep indicates the increase of interaction intensity, and the deeper the color represent the stronger the interaction. The coordinates represent its position on the genome. The nine squares are the nine chromosomes of the WWS. (c) Insect metamorphosis and its evolutionary path inferred based on comparison of 24,923 gene families from WWS and 15 other representative invertebrate genomes. The numbers on each branch correspond to the numbers of gene families that have expanded (green) and contracted (blue) in the WWS genome. All nodes in red received support levels of 90-100% and 80-89% in orange with five black dots representing time calibration by fossils and references. The estimated divergence times are included under the nodes. MYA, million years ago. The arrow and the number in orange color indicate the divergence time of the hemimetabolous separated from the holometabolan and those in purple color show the time of WWS diverge from aphids. The arrow in light blue on different order indicate the direction of insect evolution is ametabolous to hemimetabolous, then to Holometabola. Ame. is the abbreviation of ametabolous.
FIGURE 3 Methylomic analyses between females and males of WWS at the first instar nymph stage. (a) Relative CG methylation levels of different genomic regions through a WGBS analysis. The ratio was defined as the number of methylated CG to the number of total CG and was calculated using the CG on the reads that map to the defined regions. The results were derived from the data of first-instar nymphs. (b) Distribution of methylation density on the WWS whole genome. Track a: 20 linkage groups of the WWS genome. Track b: CG sequence methylation density. Track c: CHG sequence methylation density. Track d: CHH sequence methylation density. Track e: TE represents the proportion heat map of repeated sequences. Track f: Gene number density. (c) Functional classification of differentially methylated genes (DMGs) between male and female in first-instar nymphs. (d) Enrichment analysis of KEGG of DMR related genes. The bar represents the proportion of gene methylation of each pathway in the male (black) and female (red).
FIGURE 4 Juvenile hormone and ecdysone titers with gene regulation comparison between females and males. (a), (c) The change in JH and 20E titers during different development stages of male (a) and female (c) development. Males undergo through first and second instar nymphs (FM, SM), pseudo-prepupae (PP), pseudo-pupae (P), and adults (MA). Females undergo through first to second instar nymph (FF, SF) and adults (FA). (b) A heat map of genes with elevated expression in nymphs. The heat map was generated using hierarchical clustering and complete linkage of the top 500 most highly expressed orthologous genes from males and females. Different colors represent different levels of gene expression in FF, FM, SF, SM, respectively. (d) Model of molecular regulation involved in hormone metabolism pathways. Five genes are shown in red involving JH and ecdysone synthesis and degradation. The expression patterns of the genes CYP18A1 , CYP314A1 andCYP307A1 , having roles in the metabolism of ecdysone whereas the patterns of the genes JHE and CYP15A1 , having roles in the metabolism of juvenile hormone. (e) Detection of CYP18A1 target gene by PCR. Left lane is PCR amplification results of WWS cDNA whereas right lane is PCR amplification results of HT115 (CYP18A1 -L4440) bacterial solution. (f) Expression levels of CYP18A1 at 12, 24, 48 and 72 hours after RNAi treatment in the early second instar of nymphs. Control is WWS female under HT1115 treatment without target geneCYP18A1 (L4440/HT115). (g) Morphological changes of WWS female at 35, 45, 55 and 65 days were monitored after RNAi treatment. Control is WWS female under HT1115 treatment without target gene CYP18A1(L4440/HT115). CK is WWS female under natural status without any treatment. (h) Variation trend of WWS female body area at 2, 12, 22 and 42 days after RNAi treatment. Control is WWS female under HT1115 treatment without target gene CYP18A1 (L4440/HT115) and CK is WWS female under natural status without any treatment.