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Chromosomal-Level Genome Assembly of a True Bug, Aspongopus chinensis Dallas, 1851 (Hemiptera: Dinidoridae)

Abstract The true bug, Aspongopus chinensis Dallas, 1851 (Hemiptera: Dinidoridae), is a fascinating insect with prolonged diapause and medicinal properties but also a notorious pest. However, because of the lack of genomic resources, an in-depth understanding of its biological characteristics is lacking. Here, we report the first genome assembly of A. chinensis anchored to 10 pseudochromosomes, which was achieved by combining PacBio long reads and Hi-C sequencing data. This chromosome-level genome assembly was 1.55 Gb in size with a scaffold N50 of 156 Mb. The benchmarking universal single-copy ortholog (BUSCO) analysis of the assembly captured 96.6% of the BUSCO genes. A total of 686,888,052 bp of repeat sequences, 18,511 protein-coding genes, and 1,749 noncoding RNAs were annotated. By comparing the A. chinensis genome with that of 8 homologous insects and 2 model organisms, 213 rapidly evolving gene families were identified, including 83 expanded and 130 contracted gene families. The functional enrichment of Gene Ontology and KEGG pathways showed that the significantly expanded gene families were primarily involved in metabolism, immunity, detoxification, and DNA/RNA replication associated with stress responses. The data reported here shed light on the ecological adaptation of A. chinensis and further expanded our understanding of true bug evolution in general.


Introduction
Hemiptera is an insect order that is distributed across the world and contains >8,000 nonholometabolous species (Capinera 2008). Hemiptera is classified into five suborders: Sternorrhyncha (aphids, scale insects, psyllids, and whiteflies), Fulgoromorpha (planthoppers), Cicadomoirpha (leafhoppers, cicadas, and spittlebugs), Coleorrhyncha (moss bugs), and Heteroptera (true bugs) (Wang et al. 2019). True bugs are sap-sucking insects that consume food from sources ranging from animals to plants (Sparks et al. 2020). Aspongopus chinensis Dallas, 1851 (supplementary fig. S1), a true bug species mainly distributed in Eastern South Asia, is a polyphagous insect that has a prolonged diapause (Luo et al. 2012;Li et al. 2020). These two characteristics contribute to its adaptation efficiency to harsh environments. Although it is a severe threat to pumpkin and watermelon as a pest, A. chinensis has medicative and economic value; it is used in treating nephritis and gastric as well as menstrual pain (Wu et al. 2021).
In the present study, we report the first chromosomescale genome of a true bug from the Dinidoridae family, A. chinensis, which was sequenced using the Pacific Biosciences (PacBio) SMRT sequencing technology. In the genome, we annotated the repetitive sequences, proteincoding genes, and noncoding RNAs. In addition, we analyzed the gene family evolution of hemipteran clades based on the genome and annotated the function of the rapidly evolving gene families of A. chinensis. The high-quality A. chinensis genome reported in this study represents an essential genetic resource for improving researchers' understanding of the biological characteristics of the species and for conducting Hemipteran evolutionary studies.

Gene Annotation
We annotated 44.3% of the whole genome as repeat sequences, including DNA elements (6.73%), long interspersed nuclear elements (22.15%), long terminal repeats (0.03%), short interspersed nuclear elements (SINEs) (27.82%), and unclassified repeat sequences (0.77%). With a total length of 431,415,958 bp, SINEs were the richest type of repeat sequence. SINEs play crucial roles in genome evolution and modulation of gene expression (Kanhayuwa and Coutts 2016).

Gene Families and Phylogenetic Analyses
We identified 165,029 genes from 10 insect species in 18,640 orthogroups. Overall, 6,500 species-specific orthogroups (16.9% in total) were found. The gene set cluster analysis of four heteropteran species (Cimex lectularius, A. chinensis, Apolygus lucorum, and Halyomorpha halys) showed that they shared 4,122 clusters. Furthermore, A. chinensis contained 582 unique clusters (3,008 proteins), accounting for 4.94% of all clusters of the four species; these could be attributable to novel proteins and unique horizontal gene transfer ( fig.  1b). Based on 1,269 single-copy orthogroups, a phylogenetic tree was constructed that included divergence time and orthologous gene families ( fig. 1c). The phylogenetic tree and divergence time were consistent with hemipteran taxonomy and results from a previous study (Liu et al. 2021). As shown in the tree, two suborders of Hemiptera, namely Fulgoromorpha and Heteroptera, diverged from a recent common ancestor at 225-242 Ma and from Aphidomorpha approximately at 265-280 Ma. Moreover, the true bugs H. halys and A. chinensis appear to have diverged at <50 Ma, indicating that they have extraordinarily close consanguinity.
A total of 1,755 expanded and 1,113 contracted genes were identified in 83 significantly expanded and 130 contracted gene families, respectively. The functional annotation of the 83 significantly expanded gene families indicated their association with metabolism, environmental adaptation, digestion, transport, and immune and detoxification systems ( fig. 2a). Metabolic pathways were significantly enriched in A. chinensis; these are likely associated with overwintering behavior and metamorphosis (Klowden 2013). In addition, dynamic changes in nutrients are generally used by insects to regulate the diapause period (Hahn and Denlinger 2011).
Further analyses showed that the expanded gene families enriched in GO ( fig. 2b) and KEGG (fig. 2c) primarily functioned in metamorphosis, immunity, DNA/RNA replication, detoxification, and metabolism, which may explain the mechanism of the broad phytophagy of A. chinensis as well as its adaptation to adverse environmental conditions. One specifically identified KEGG pathway, retinol metabolism, is associated with color vision and therefore essential for habitat exploration and foraging (Lebhardt and Desplan 2017). Furthermore, metabolic pathways (including carbohydrate, lipid, cofactor and vitamin, ascorbate and aldarate, and energy metabolisms), are significantly enriched in diapausic insects compared with non-diapausic A. chinensis groups (Qi et al. 2015;Chen et al. 2017;Wu et al. 2021). In the KEGG clustered pathways, genes involved in the HIF-1 signaling pathway are significantly expressed in diapause-destined pupal brains (Lin et al. 2016). Overall, these analyses provide further insights into the genomics of A. chinensis, particularly in terms of its feeding traits and environmental adaptation.

Sample Collection and Sequencing
Aspongopus chinensis samples were collected from Kaili City, Guizhou Province, China (26 39 0 16.65 00 N, 107 47 0 18.73 00 E) and subsequently reared for at least three generations under controlled conditions (25 C, 80% relative humidity, and a 14:10-h light:dark photoperiod) at the Institute of Entomology, Guizhou University. To survey the genome size and heterozygous ratio, we constructed a 350-bp insert-size DNA library for Illumina sequencing on the HiSeq NovaSeq 6000 platform, a 20-kb insert-size library sequencing using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, CA, USA), and a 150 paired-end RNA library using TRIzol Reagent. We filtered the raw reads with short lengths (<50 bp) and low-quality values (<0.8) and then removed the adapter sequence consistencies.

Supplementary Material
Supplementary data are available at Genome Biology and Evolution online.