3.4 Identification of DEGs between winged and wingless M. dirhodum
All aphids are born through viviparous reproduction with wing primordia, but they degenerate by the second instar in the unwinged morph (Ding et al., 2017). In the winged individuals, the wing primordia continue to slowly develop from first- to third-instar nymphs and then rapidly grow in the fourth instar. In M. dirhodum , winged and wingless individuals can be distinguished from third-instar nymphs to adults under a microscope (Ishikawa et al., 2008, 2013). To identify potential genes related to wing dimorphism and development, RNA-seq was performed between populations of winged and wingless M. dirhodum in third- and fourth-instar nymphs and adults using our assembled genome as a reference.
As a result, 4641 DEGs were detected between the wingless (WW) and winged (WY) third-instar nymphs (WW3/WY3), including 2114 upregulated and 2527 downregulated genes in the wingless nymphs, compared to the winged individuals (Fig. 4A, Table S4). A total of 4349 DEGs were detected between the wingless and winged fourth-instar nymphs (WW4/WY4), including 1936 upregulated and 2413 downregulated genes in the wingless nymphs, compared to the winged individuals (Fig. 4B, Table S4). In addition, 4375 DEGs were detected between the wingless and winged adults (WWA/WYA), including 1877 upregulated and 2498 downregulated genes in the wingless adults, compared to the winged individuals (Fig. 4C, Table S4). Among them, 3638 genes were differentially expressed in both the WW3/WY3 and WW4/WY4 groups, 3362 genes were differentially expressed in both the WW3/WY3 and WWA/WYA groups, 3433 genes were differentially expressed in both the WW4/WY4 and WYA/WWA groups, and 3050 genes were differentially expressed in all three comparison groups (WW3/WY3, WW4/WY4 and WWA/WYA) (Fig. 4D, Table S4).
Juvenile hormone or ecdysone signals, c-Jun NH2-terminal kinases (JNK), and insulin/insulin-like growth factor signaling (IIS) pathways have been reported to contribute to the regulation of wing dimorphism in many insects (Xu et al., 2015, 2017; Zhang et al., 2019). Moreover, Wnt2, Fng (fringe), Uba1 (ubiquitin-activating enzyme E1), Hh (hedgehog), Foxo, Dpp (decapentaplegic), Brk (Brinker), Ap (alar process), Dll (distal-less), Hth (helix-turn-helix), Tsh (thyroid-stimulating hormone), Nub (nubbin), Scr (sex combs reduced), Antp (antennapedia), Ubx (ultrathorax), Asc, Srf (serum response factor) and Fl (flugellos) have been also reported to play important roles in regulating wing polyphenism or development (Zhang et al., 2019). All the DEGs annotated as these genes were selected and are listed in Tables 2, 3 and 4, including insulin receptor substrate, insulin-like receptor, ecdysone receptor, broad-complex core protein, and Foxo. In addition, many genes related to muscle composition, energy metabolism and reproduction were also identified between the winged and wingless aphids.