3.4 ∣ Hormone analysis
Hormone titers of WWS males and females have been determined from nymphs
to adults (Figure 4 a, c). Unlike previous results reported from other
insect species, our study suggests that ecdysone (20E) antagonizes
juvenile hormone (JH) during WWS metamorphosis. Throughout the life
cycle, JH titers in WWS females and males appeared to display a downward
trend, with minimal JH levels detectable at the adult stage (Figure 4 a,
c). Ecdysone was much higher than JH in males (holometabola). Even in
females (hemimetabola), ecdysone was much higher than JH except in early
first larval stage (Figure 4 a, c; Supplementary Figure 10). During the
second larval and adult stages, neoteny females also showed much higher
ecdysone. Our results is different from previous studies that showed JH
mediates neoteny in insects (AZinna et al.,
2018; Stillwell et al., 2010),
indicating that there are different mechanisms in insect metamorphosis
regulation.
In addition to the comparative genomic analyses, we performed a
transcriptomic analysis of WWS-based RNA-seq data from nymphs of both
sexes (Figure 4b). Differentially expressed genes were more pronounced
in males at different stages than females when the second instar nymphs
were compared with larvae (Figure 4b). This may reflect the differences
in developmental regulation between males and females. A hierarchical
clustering analysis of the differentially expressed genes related to
hormone metabolism also showed significant differences between males and
females, particularly at the second instars. In general, genes involved
in ecdysone and JH synthesis were expressed at higher levels in second
instar females, whereas genes involved in ecdysone and JH degradation
were expressed at lower levels. For example, CYP314A1 (shade) andCYP307A1 (spook) , which encode cytochrome P450 enzymes involved
in ecdysone synthesis; and CYP15A1, which encodes an enzyme
involved in synthesizing JHIII from methyl farnesoate
(Helvig, Koener, Unnithan, & Feyereisen,
2004); were expressed at higher levels in second instar females. On the
other hand, a gene encoding a JH esterase (JHE), which plays a role in
breaking down JH; and CYP18A1 , which encodes an enzyme to break
down ecdysone; were expressed at lower levels in the second instar
females. The expression patterns of genes related to hormone metabolism
were consistent with a significant drop in JH in second instar males
(Figure 4b, d) (Helvig et al., 2004)
(Figure 4b, d). The overall higher expression levels of hormone
synthesis genes in the second-instar than in first-instar nymphs suggest
that both JH and 20E production are ramped up in the second instars.
Based on their expression patterns and putative roles in hormone
metabolism, the five putative genes CYP314A1 , CYP307A1 ,CYP15A1 , CYP18A1 , and the JHE-encoding gene were selected
for follow-up RNAi studies to provide direct evidence for their roles in
hormone metabolism. Interference effect of RNAi on gene expression was
observed between 24 to 72 hours (72h) (Figure 4e, f). SilencingCYP18A1 slowed down growth of females (Figure 4g, h), suggesting
that CYP18A1 depletion interfered with growth of WWS larvae.