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.