4 ∣ DISCUSSION
Here we report a high-quality genome sequence assembled from an important scale insect species. Among the 14,020 protein-encoding genes predicted from this genome sequence, 6,189 genes were unique and 5,599 of them were supported by transcripts, indicating that these genes encode real products (Supplementary Figure 11). Previous molecular phylogenetic studies only used a number of nuclear and mitochondrial genes to construct the evolutionary tree, which might have strong bias in data sampling and could not well reflect the evolutionary relationship between different species in insects (Misof et al., 2014). The molecular evolution tree based on whole genome information can avoid some of the problems (Figure 2c). The large number of unique genes discovered from this study provides a useful resource for future WWS studies and should also be useful for future comparative studies with other organisms.
Expansion and reduction of genes are usually associated with adaptation of species to specific ecological requirements in their respective habitats (Harris & Hofmann, 2004; Lespinet et al., 2002). Parasitic insect species typically display expanded gene families that allow for feeding of the hosts (Oliva et al., 2015; Rays et al., 2013; Zhao et al., 2015). Plant pathogens have expanded gene families that function in cell wall degradation to facilitate invasion to their host plants (Morales-Cruz et al., 2015). Compared to other insect genomes, there are several categories of genes that underwent significant expansion in the WWS genome. One of these categories corresponds to fatty acid metabolism, including fatty acid synthases and low-density lipoprotein. UDP-glucuronosyltransferases, involved in removing xenobiotics, are also expanded significantly, which may have allowed the insect to eliminate endogenous and exotic toxic chemicals.
One of the striking features of the WWS genome is its unique DNA methylation pattern with the highest methylation level (Supplementary Table 18) in the class of hexapoda that has been reported so far (Phalke et al., 2009; Regev et al., 1998; Xiang et al., 2010). The high methylation of the WWS genome may have allowed the insect for high phenotypic plasticity to cope with environmental challenges since this females insect species is immobile at the adult stage, and as such, hard to evade harsh environmental conditions.
Two families of hormones, the ecdysteroids and the juvenile hormones, control molting and metamorphosis during postembryonic life (Gilbert, 1994). Ecdysone induces the production of a new cuticle, whereas JH regulates the character of molting (Truman & Riddiford, 1999). During molts, JH levels are high, but fall dramatically to practically undetectable in the pre-adult stage (Belles, 2011). JH represses metamorphosis, and its downregulation is required for metamorphosis to occur (Riddiford, 2008). In our study, 20E titers in male development shows five distinct peaks, while females only display two, consistent with the idea the male development is more complex. Remarkably, females exhibit a pronounced peak of JH in the 1ststage nymph, with a corresponding drop in ecdysone. As such, the determinant of female development appears to occur early in development, since the JH titers are dramatically higher in FF compared to FM samples and the relationship between JH and ecdysone is reversed in males (Figure 4a-c). In fact, the female 1ststage nymph is the only time in either of the sexes where JH eclipses absolute ecdysone concentrations. Therefore, we propose that the key developmental events that lead to female development are established already in 1st stage nymphs. Later in development, the additional ecdysone peaks observed in males are likely associated with the development of morphological features specific to males.
Insect metamorphosis is a fascinating adaptation that allows the transformation of nymphs into reproductive adults. Ancestral insect species did not undergo metamorphosis and there are still some extant species that lack metamorphosis or only undergo partial metamorphosis (Truman & Riddiford, 1999). Both hemimetamorphosis and complete metamorphosis exist in the male and female of white wax insect, respectively (Supplementary Figs. 2). This suggests that WWS represents a transitional position between hemimetamorphosis and holometabolic insects. The main benefits of sexually dimorphic metamorphosis in WWS are likely the ability of larvae and adults to utilize different habitats and food resources, and this may have serves as a force to accelerate the evolution of rapid life cycles and enhance their chance for survival effectively.
From this respect, WWS represents a fascinating model not only to study the adaptive mechanisms of plant-sap feeding parasites but also to examine the evolutionary relationships between hemimetaboly and holometaboly in different insect orders. The phylogenetic status, evolution of WWS gene families presented here provide a key case and framework for furtherly understanding the evolution of insect metamorphosis and functional herbivory adaption.
Our results provided comprehensive genome and epigenome resource of scale insects and shed new insights into the different evolution paths of metamorphosis between males and females within the same species. It represents a conceptual framework for future studies that examine the evolution of hexapod metamorphosis.