4 DISCUSSION
M. dirhodum is an important pest of wheat and other cereals worldwide. Previous studies of this species have mainly focused on occurrence regularity (Honek et al., 2018), control methods (Cambier et al., 2001; Chopa et al., 2012) and reproductive strategies (Wratten, 1977), whereas more in-depth studies are limited due to the lack of high-quality genome data. Therefore, we used PacBio long HiFi reads and Hi-C technology to produce a chromosomal-level genome for M. dirhodum . This is the first high-quality chromosome-level genome ofM. dirhodum , which will be very helpful for cloning, functional verification and evolutionary analysis of genes in this important species or even in other hemipteran insects.
The k -mer analysis showed that the M. dirhodum genome harbored a moderate level of heterozygosity and a high level of repetitive sequence content, which is similar to other Aphidinae insects with low or moderate level heterozygosity (Jiang et al., 2019; Chen et al., 2019). BUSCO assessment revealed that 96.9% of the complete BUSCOs could be found in the current assembled M. dirhodum genome. This percentage is higher than those in the genomes of some other species, such as S. miscanthi (90.2%) (Jiang et al., 2019), R. maidis (94.5%) (Chen et al., 2019), A. pisum (93.5%) (International Aphid Genomics Consortium., 2010) and E. lanigerum(96.8%) (Mathers et al., 2021). Considering the moderate level of heterozygosity and the high-level repetitiveness of the genome, the current result represents a high-quality genome assembly of M. dirhodum . Nine chromosomes were finally obtained after Hi-C-assisted assembly, supporting a 2n = 18 karyotype for M. dirhodum, which is identical to S. miscanthi (Jiang et al., 2019). Furthermore, a total of 18,003 protein-coding genes were predicted in the genome ofM. dirhodum , which was comparable to that of several other Aphidinae species, such as 16,006 protein-coding genes in S. miscanthi (Jiang et al., 2019) and 19,097 protein-coding genes in D. noxia (Nicholson et al., 2015), but less than R. maidis (Chen et al., 2019), A. pisum(International Aphid Genomics Consortium., 2010), M. persicae(Jiang et al., 2013) and E. lanigerum (Mathers et al., 2021), which have 26,286, 36,195, 23,910 and 28,186 protein-coding genes, respectively.
Wing polymorphism is an evolutionarily successful feature in a wide variety of insect species, including Hemiptera, Coleoptera, Hymenoptera, Orthoptera, Diptera, Lepidoptera, Isoptera, Psocoptera and Dermaptera (Xu et al., 2017; Zhang et al., 2019). Insect wing polymorphisms have been widely studied, and some progress has been made, especially in the rice plant hopper Nilaparvata lugens , in which two putative insulin receptors (InRs), InR1 and InR2, were identified. Interestingly, InR1 and InR2 play fully opposite roles in wing morph determination by regulating the activity of Foxo. In detail, knockdown of InR2 at the nymph stage led to a strong trend toward long-winged adults, whereas dysfunction of InR1 resulted in the development of short-winged adults (Xu et al., 2015). In the present study, two transcripts annotated as insulin receptors (insulin-like receptors and insulin-like peptide receptors) were identified in M. dirhodum , which is consistent with the annotation of the A. pisum genome (International Aphid Genomics Consortium., 2010). Comparative transcriptome analysis showed that the insulin-like receptor was downregulated in third-instarM. dirhodum wingless nymphs and that the insulin-like peptide receptor was upregulated in fourth-instar wingless nymphs compared to winged individuals. In addition, one transcript annotated as insulin receptor substrate 1 was downregulated in both third- and fourth-instar wingless nymphs, and one transcript annotated as insulin gene enhancer protein isl-1 was upregulated in both third-instar and fourth-instar wingless nymphs compared to the winged individuals. No DEGs associated with the insulin signaling pathway were identified between the wingless and winged M. dirhodum adults. These results suggest that insulin signaling may play an important role in the early wing dimorphism ofM. dirhodum . Foxo was identified to be upregulated in winglessM. dirhodum in third- and fourth-instar nymphs and adults, which is another important factor in the regulation of wing dimorphism. Vellichirammal et al. (2017) revealed that ecdysone signaling plays a critical role in transgenerational plasticity in wing dimorphism inA. pisum . Their results showed that more winged offspring would be produced by injection of ecdysone or its analog, while fewer winged offspring were produced by knockdown of EcR or treatment with an EcR antagonist. Our results showed that EcR is downregulated in winglessM. dirhodum from third-instar nymphs to adults compared to winged nymphs, suggesting that the decrease in ecdysone is not conducive to the production of winged individuals. Moreover, DEGs annotated as juvenile hormone epoxide hydrolase, brc, FTZ-F1, Wnt, Dpp, Ubx, Srf and Fl were also detected between the wingless and winged M. dirhodum . These findings provide a valuable reference for revealing the mechanism of wing dimorphism.