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.