Genome-Wide Comparative Analysis of the Cytochrome P450 Monooxygenase Family in 19 Aphid Species and Their Expression Analysis in 4 Cereal Crop Aphids

Abstract

Cytochrome P450 monooxygenases (CYP450s) play a variety of physiological roles, including pesticide resistance, plant allelochemical detoxification, and hormone metabolism catalysis. However, limited information is available on the classification and expression profiles of the CYP450 gene family in aphid species. This is the first study to identify the cytochrome P450 gene family in 19 aphid species at the whole genome level. A total of 1100 CYP450 genes were identified in 19 aphid species. Three hundred CYP450 genes belonged to six cereal crop aphid species, which were further classified into four subfamilies according to the phylogenetic relationship. The conserved motifs, exon-intron structures, and genomic organization of the same subfamilies were similar. Predictions of subcellular localization revealed that the endoplasmic reticulum harbored the majority of CYP450 proteins. In Sitobion avenae and Rhopalosiphum maidis, the increase in the CYP450 gene was primarily caused by segmental duplication events. However, only tandem duplication occurred in the CYP450 gene family of Diuraphis noxia, Rhopalosiphum padi, Schizaphis graminum, and Sitobion miscanthi. Synteny analysis found three continuous colinear CYP450 gene pairs among six cereal crop aphid species. Furthermore, we obtained the expression profiles of four cereal crop aphids, including R. padi, D. noxia, S. graminum, and S. avenae. Differential expression analysis provided growth stage specificity genes, tissue specificity genes, organ specificity genes and some detoxification metabolic genes among these four cereal crop aphids. Meanwhile, their expression patterns were showed. The related functions and pathways of CYP450s were revealed by GO and KEGG enrichment analysis. Above all, we picked the differentially expressed CYP450 genes from all of the differentially expressed genes (DEGs). These differentially expressed CYP450 genes provided some new potential candidates for aphid control and management. This work establishes the foundation for further investigations into the regulatory functions of the CYP450 gene family in aphid species and beyond.

1. Introduction

Aphids, one of the most significant pests in global agriculture, cause harm to crops and other plants through sucking phloem sap, spreading plant viruses, and reducing photosynthetic efficiency. Out of approximately 5000 species of aphids, approximately 100 have been recognized as important agricultural pests [1]. Pea aphid (Acyrthosiphon pisum) feeds on legume species across the globe, including forage crops like pea, clover, alfalfa, and broad beans [2]. Aphis craccivora (Koch) feeds on legume plants, and carries bean leaf roll virus and broad bean necrotic yellows virus [3]. Soybean aphid (Aphis glycines), sucks liquid from leaves and stems, causing direct damage to soybean plants [4]. Melon/cotton aphid (Aphis gossypii Glover) parasitizes many vegetable crops, especially Cucurbitaceae, causing serious direct and indirect harm to host plants through sucking and serving as a viral vector [5,6]. Foxglove aphid (Aulacorthum solani) reduces soybean yield by spreading viruses and sucking in leaf sap directly [7]. Cinara cedri feeds on cedar branches, secretes a large amount of honeydew, and pollutes the lower branches [8]. Daktulphophora vitifoliae (Fitch) mainly sucks the juice of grapes [9]. The woolly apple aphid (WAA), Eriosma lanigerum, inserts probe mouthpieces into the bark and feeds on the sap from the phloem, causing serious damage [10]. The sugarcane aphid, Melanaphis sacchari (Zehntner), feeds on the phloem of plants, leading to chlorosis, leaf curling/withering, and necrosis. [11]. The banana aphids (Pentalonia nigronervosa Coquerel) carry viruses that cause severe disease and significant yield losses in bananas [1]. Myzus persicae (Sulzer), the green peach aphid, can spread hundreds of plant viral diseases such as potato leaf rolling disease and sugar beet yellow flower web disease [12]. The yellow sugarcane aphid (Sipha flava) mainly infests corn, rice, sorghum, and sugarcane, and parasitizes lawn, pasture grasses, and some sedges [13]. The black cherry aphid (Myzus ceras) can result in pseudogall development, shoot deformation, and leaf curling [14]. In addition, the English grain aphid (Sitobion avenae Fabricius), the greenbug (Schizaphis graminam), Sitobion miscanthi, and Rhopalosiphum padi are a widely distributed pest in wheat and other cereal crops, as they spread different plant viruses (such the barley yellow dwarf virus) and extract phloem sap directly, causing serious yield losses [15,16,17]. Numerous harmful maize viruses, such as the yellow dwarf virus of maize, the barley yellow dwarf virus, the mosaic virus of sugarcane, and the mosaic virus of cucumber are spread by the corn leaf aphid (Rhopalosiphum madis Fitch) [18]. Diuraphis noxia (Kurdjumov) mainly threatens the production of wheat and barley in Australia, while it also slightly damages rye, triticale, and oats [19]. However, chemical insecticides have been, and will continue to be, widely used for controlling aphids, which will lead to the evolution of resistance in aphid species.

In order to resist the toxicity of insecticides, insects have evolved corresponding strategies to address this challenge, in which detoxification metabolic enzymes play an important role. Common detoxification metabolic enzymes mainly include glutathione S-transferases (GSTs), esterases (ESTs), and cytochrome P450 monooxygenases (CYP450s) [20]. Cytochrome P450 is a family of self-oxidizing heme proteins that are ubiquitous in animals, plants, and microorganisms. The cytochrome P450 monooxygenase is named after its main component, the P450 protein, which binds to CO and exhibits a characteristic light absorption peak at 450 nm. The diversity of P450 protein types and the overlap of substrates enable the P450 enzyme system to catalyze various types of reactions. It is primarily involved in the metabolism of foreign drugs like pesticides and plant secondary metabolites in insects, as well as the creation and breakdown of endogenous compounds like pheromones and ecdysone [21]. Overall, P450 can catalyze thousands of reactions, even exhibiting multiple types of reactions on substrates with similar chemical structures. The P450 catalytic reaction process involves multiple steps, and its mechanism of action is diverse. A typical reaction involves reducing molecular oxygen through an electron transfer system and adding one of its oxygen atoms to the substrate, which requires nicotinamide adenine dinucleotide phosphate NADPH. With the quick advancement of high-throughput sequencing technologies in recent years, more than 1000 P450 genes have been reported up to now. It is generally believed that the insect P450s gene family consists of four branching clusters, namely CYP2 clan, CYP3 clan, CYP4 clan, and Mito clan. Among the 37 animal P450 families, 16 families are found only in mammals, while the other 21 families are distributed in insects (6), mollusks (2), and Caenorhabditis elegans (13).

The role of the P450 enzyme system in insects involves processes such as growth, development, and feeding. It is implicated not only in insect resistance to insecticides, but also in the synthesis and metabolism of hormones in insects. Research has shown that the CYP18A1 protein of Chironomus riparius larvae has the function of degrading the molting hormone [22]. The CYP6DE1 protein can catalyze α-pinene metabolism to produce aggregation pheromones in Dendroctonus Ponderosae [23]. The detoxification activity can be strengthened by overexpressing CYP450s and accumulating or altering the structure of detoxifying proteins [24,25]. Additionally, studies have demonstrated that broad substrate specificity, genetic diversity, and catalytic plasticity of P450s contributed significantly to pesticide resistance. In the CYP450 gene family, insecticide resistance is mostly correlated with genes in the CYP4 and CYP6 families [26]. The CYP6AL1 protein of Aedes aegypti larvae is engaged in the process of detoxifying harmful exogenous plant compounds [27]. CYP6G1 is the main factor causing Drosophila melanogaster to develop resistance to dichlorodiphenyltrichloroethane (DDT) [28]. Studies have demonstrated that imidacloprid resistance in Bemisia tabaci can result from CYP6CM1 overexpression [29]. Imidacloprid resistance was increased in Drosophila expressing the CYPC6Y9, CYP6CY22, CYP6CY18, and CYP6D subfamily genes (>5 fold) [30]. Melon/cotton aphids develop a metabolic resistance to dinotefuran due to the overexpression of three cytochrome P450 genes, CYP6CY14, CYP6CY22, and CYP6UN1 [31]. The CYP380C6 of green peach aphid facilitated their adaptation to indole-glucoside-mediated plant defense [32]. CYP6DB3 participates in the resistance of Bemisia tabacito to thiamethoxam and imidacloprid [33]. It was reported that CYP321B1 participated in insecticide detoxification of Spodoptera litura [34]. In Megalurothrips usitatus Bagnall, RNA interference of the CYP450 gene resulted in reducing pesticide resistance [35].

The failure of numerous economically significant pest control efforts and the comprehension of the role played by the P450 enzyme system in insect pest control have spurred research into insect P450. However, due to the inherent characteristics of insect P450, biochemical methods are limited in their ability to study its structure and function. In this study, we identified CYP450 family genes in 19 aphid species using the genome-search method based on the available aphid genome. The genomic organizations, phylogenetic relationships and conserved domain of the CYP450 family genes were investigated among 19 aphid species. Differential expression analysis provided growth stage specificity CYP450s, tissue specificity CYP450s, organ specificity CYP450s, and some detoxification metabolic CYP450s among these four cereal crop aphids, which provided some new potential candidates for aphid control and management. This work established the foundation for further investigations into the regulatory functions of the CYP450 gene family in aphid species and beyond.

2. Results

2.1. Genome-Wide Identification of CYP450 Genes in 19 Aphid Species

A total of 1100 CYP450 genes were identified in 19 aphid species (Table S1). D. vitifoliae had the largest number of CYP450 genes (78), followed by A. solani (76) and S. avenae (74). There were only 19 CYP450 genes in S. miscanthi, which was the specie with the least amount of CYP450 genes (Figure 1A). The length of CYP450 genes ranged from 141 bp (DV3001928-RA from D. vitifoliae) to 101,993 bp (Sgrm016186.1 from S. graminum) according to sequence characteristic analysis (Figure S1). The CDS length of CYP450 genes ranged from 141 bp (DV3001928-RA from D. vitifoliae) to 4623 (Smis013654.1 from S. miscanthi) (Figure 1B). The largest- and smallest-sized of amino acid sequences were 1541 aa and 46 aa, respectively (Figure S2). The average molecular weight of CYP450 proteins was 94.65 kDa, with a range from 5.31 kDa (DV3001928-RA from D. vitifoliae) to 167.96 kDa (Smis013654.1 from S. miscanthi) (Figure 1C). As for each aphid specie, we found that S. flava had the largest average value of the gene length (8112 bp), CDS length (1554 bp), protein size (517 aa), and molecular weight (59.47 kDa). However, A. solani had the lowest average value of gene length (1959 bp), CDS length (878 bp), protein size (292 aa), and molecular weight (33.43 kDa). The predicted isoelectric points (pI) of CYP450 proteins varied from 3.72 (jg703.t1 from E. lanigerum) to 10.72 (jg6703.t1 from E.lanigerum) (Figure S3). A. solani had the lowest average value of pI (7.84), but M. sacchari had the largest average value of pI (8.93). The majority of CYP450 proteins were located in the endoplasmic reticulum (453 out of 1100), cytoplasm (338 out of 1100), and plasma membrane (140 out of 1100) (Figure 1D). Specifically for each specie, most of CYP450 proteins were located in the endoplasmic reticulum and cytoplasm in 17 aphid species. Moreover, the number of CYP450 genes in the endoplasmic reticulum is greater than that in the cytoplasm in 13 aphid species. The number of CYP450 genes located in the endoplasmic reticulum is less than that in the cytoplasm among four aphid species (A. craccivora, A. solani, D.vitifoliae, and E. lanigerum). However, most of CYP450 proteins were located in the endoplasmic reticulum and plasma membrane in R. maidis and S. graminum. All CYP450 proteins had a projected grand average of hydropathicity (GRAVY) value of negative, which suggested that they were hydrophilic by nature.

We further compared the gene number and sequence characteristics of CYP450 genes among the cereal crop aphid species, including D. noxia, R. maidis, R. padi, S. graminum, S. miscanthi, and S. avenae. There were 300 gene members among these 6 cereal crop aphid species (Table S2). S. avenae had the largest number of CYP450 genes (74), and S. miscanthi had the least number of CYP450 genes (19) (Figure 1A). According to their chromosomal locations, the predicted CYP450 genes were given names like DnoxCYP1. Sequence characteristic analysis revealed that the length of CYP450 genes ranged from 566 bp (DnoxCYP12 from D. noxia) to 101,993 bp (SgrmCYP33 from S. graminum) (Figure S1). The CDS length of CYP450 genes ranged from 294 bp (SaveCYP10 from S. avenae) to 4623 bp (SmisCYP17 from S. miscanthi) (Figure 1B). The largest- and smallest-sized amino acid sequences were 1541 aa (SmisCYP17) and 98 aa (SaveCYP10), respectively (Figure S2). The molecular weight of CYP450 proteins ranged from 11.00 kDa (SaveCYP10 from S. avenae) to 167.96 kDa (SmisCYP17 from S. miscanthi) with the average value of 94.65 kDa (Figure 1C). As for each cereal crop aphid specie, we found that S. miscanthi had the largest average value of CDS length (1711 bp), protein size (569 aa), and molecular weight (64.57kDa). However, D. noxia had the lowest average value of the CDS length (1366 bp), protein size (444 aa), and molecular weight (51.08 kDa). The pI of CYP450 proteins varied from 4.49 (DnoxCYP42 from D. noxia) to 10.33 (DnoxCYP13 from D. noxia) (Figure S3). D. noxia had the lowest average value of pI (8.16), but S. miscanthi had the largest average value of pI (8.51). Subcellular localization predictions revealed that the majority of CYP450 proteins were located in the endoplasmic reticulum (136 out of 300), cytoplasm (72 out of 300), and plasma membrane (50 out of 300) (Figure 1D). The predicted GRAVY value of these 300 CYP450 proteins was also minus, showing that they were hydrophilic in nature.

2.2. Chromosome Localizations of CYP450 Genes among 6 Cereal Crop Aphid Species

Chromosome localizations found some variations among six cereal crop aphid species. There were 39 scaffolds with CYP450 genes detected in R. padi, which was the specie which owned the largest number of chromosomes with CYP450 genes, while R. maidis had only 4 scaffolds with CYP450 genes (Figure 2). We found 46 CYP450 genes distributed on 4 out of 220 scaffolds of R. maidis, of which NC_040878.1 possessed the most abundant gene number of CYP450 genes (24), followed by NC_040879.1 (9) and NC_040880.1 (9), and NC_040877.1 (4) (Figure 2A). In total, 19 CYP450 genes were distributed on 7 out of 655 scaffolds of S. miscanthi, of which Lachesis_group2 possessed the most abundant gene number of CYP450 genes (9), followed by Lachesis_group0 (3) and Lachesis_group8 (3), and Lachesis_group1(1),ctg575_wtdbg_pilon(1), ctg646_wtdbg_pilon(1), and ctg662_wtdbg_pilon (1) (Figure 2B). There were 74 CYP450 genes distributed on 23 out of 326 scaffolds of S. avenae, of which ptg000011l possessed the most abundant gene number with 8 CYP450 genes and ptg000011l with 1 CYP450 gene (Figure 2C). Results showed that 53 CYP450 genes were distributed on 37 out of 5637 scaffolds of D. noxia, of which NW_015368409.1 and NW_015368405.1 possessed the most abundant gene number with 4 CYP450 genes, and 26 scaffolds had 1 CYP450 gene (Figure S4). In total, 57 CYP450 genes were distributed on 39 out of 2172 scaffolds of R. padi, of which scf7180000008163.830738 possessed the most abundant gene number with 4 CYP450 genes, and 24 scaffolds had 1 CYP450 gene (Figure S5). Similarly, 51 CYP450 genes distributed on 33 out of 7859 scaffolds of S. graminum, of which QEWZ01001422.1 and QEWZ01000247.1 possessed the most abundant gene number with 5 CYP450 genes, and 22 scaffolds had 1 CYP450 gene (Figure S6).

2.3. Phylogenetic Relationship Analysis of CYP450 Genes among Six Cereal Crop Aphids

Using the full-length protein sequences of the 300 discovered CYP450 genes, a neighbor-joining (NJ) tree was built in order to ascertain the evolutionary pattern and phylogenetic relationship of CYP450 genes among six cereal crop aphid species. These 300 CYP450 genes contained 53 DnoxCYPs, 46 RmaiCYPs, 57 RpadCYPs, 51 SgrmCYP18s, 19 SmisCYP13s, and 74 SaveCYPs (Table S1). All CYP450 genes among six cereal crop aphids were classified into four subfamilies, CYP I, CYP III, CYP III, and CYP IV (Figure 3 and Figure 4A). In detail, the CYP I subfamily included 116 members (20 DnoxCYPs, 16 RmaiCYPs, 22 RpadCYPs, 18 SgrmCYPs, 12 SmisCYPs, and 28 SaveCYPs), the CYP II subfamily contained 92 members (19 DnoxCYPs, 14 RmaiCYPs, 16 RpadCYPs, 15 SgrmCYPs, 2 SmisCYPs, and 26 SaveCYPs), the CYP III subfamily contained 37 members (7 DnoxCYPs, 6 RmaiCYPs, 7 RpadCYPs, 6 SgrmCYPs, 2 SmisCYPs, and 9 SaveCYPs), as well as the remaining 55 members belonged to the CYP IV subfamily (7 DnoxCYPs, 10 RmaiCYPs, 12 RpadCYPs, 12 SgrmCYPs, 3 SmisCYPs, and 11 SaveCYPs), respectively. Additionally, we found that RmaiCYPs, RpadCYPs, and SmisCYPs always clustered together on the same clade, indicating R. maidis, R. padi, and S. graminum had a closer genetic relationship. Similarly, DnoxCYPs, SmisCYPs, and SaveCYPs always clustered together on the same clade, indicating that D. noxia, S. miscanthi, and S. avenae had a closer genetic relationship.

2.4. Gene Structure and Conserved Motif Analysis of CYP450 Genes among Six Cereal Crop Aphids

Results showed that all the identified CYP450 genes among six cereal crop aphids possessed the conserved cytochrome P450 superfamily domain (Figure 4C). A total of ten motifs were identified, and the number and type of motifs among the four CYP450 subfamilies were quite different (Figure 4B and Figure S7). In the CYP I subfamily, almost all members (87 out of 116) had all of the ten motifs. In total, 69 CYP450 genes (69 out of 92) in the CYP II subfamily had eight motifs without motif 5 and motif 8, which was unique for the CYP II subfamily. In the CYP III subfamily, eighteen CYP450 genes had seven motifs, eight CYP450 genes had six motifs, and five CYP450 genes had five motifs. The members of the CYP III subfamily lacked motif 5. In total, 29 CYP450 genes (29 out of 55) belonged to the CYP IV subfamily and had 5 motifs, and the remaining CYP450 genes of the CYP IV subfamily had 2~6 motifs. The members of the CYP IV subfamily lacked motif 4 and motif 7.

The exon-intron structure is an important component of gene evolution that offers essential clues for its functional diversity. The results revealed that the number of exons varied from 1 to 34, with 215 CYP450s possessing more than five exons and only DnoxCYP1 and SaveCYP28 having one exon (Figure 4D). Among all the CYP450s, SmisCYP17 had 33 introns, DnoxCYP1 and SaveCYP28 had none, and the number of introns varied from 0 to 33. In total, 155 CYP450 genes had both the 5′ and 3′ UTR regions, while 23 CYP450 genes only had one UTR region (5′ or 3′ UTR region), and 122 CYP450 genes had no UTR region at all. It was clear that members of the same group had conserved motif and intron-exon structures in common, which supported the classification and evolutionary relationship of the identified CYP450s.

2.5. Gene Duplication and Synteny Analysis of CYP450 Genes among Six Cereal Crop Aphid Species

The chromosome reduplication process makes each gene possess homoeologous copies. We compared expansion events of the CYP450 gene family, including gene duplications and tandem duplications, among six cereal crop aphids. Results found that CYP450 gene family expansion events of R. maidis and S. avenae possessed both gene duplications and tandem duplications, while the remaining four cereal crop aphid species only had tandem duplications. A total of 24 duplication events of the CYP450 gene family occurred in the whole genome of R. maidis, including 7 tandem duplications and 17 segmental duplications (Figure 5A and Table S3). In total, 73 duplication events of the CYP450 gene family were identified in the whole genome of S. avenae, including 14 tandem duplications and 59 segmental duplication events (Figure S8 and Table S3). Segmental duplication events were the main reason for the expansion of CYP450 genes in S. avenae and R. maidis. There were 14, 10, 7, and 2 tandem duplications of the CYP450 gene family which occurred in D. noxia, R. padi, S. graminum, and S. miscanthi, respectively (Table S3).

In order to thoroughly examine the homology of the CYP450 family members among these six cereal crop aphid species, a colinear map of CYP450 genes was drawn. A total of 127 pairs of colinear genes were discovered (Figure 5B). There were 33, 32, 34, 22, and 6 collinearity events between S. graminum and S. avenae, S. avenae and R. padi, R. padi and R. maidis, R. maidis and D. noxia, and D. noxia and S. miscanthi, respectively (Table S4). It was evident that R. padi and R. maidis had the greatest number of collinear genes, suggesting that CYP450 genes in these two aphid species may have shared an ancestral sequence and functioned similarly. Interestingly, three continuous colinear gene pairs were found among six cereal crop aphid species, which were SgrmCYP42-SaveCYP36-RpadCYP44-RmaiCYP2-DnoxCYP6-SmisCYP5, SgrmCYP32-SaveCYP48-RpadCYP3-RmaiCYP38-DnoxCYP28-SmisCYP8, and SgrmCYP14-SaveCYP54-RpadCYP37- RmaiCYP29-DnoxCYP39-SmisCYP17. These CYP450 genes were very conserved among six cereal crop aphid species, which developed with the aforementioned homologous genes prior to the differentiation of these species.

To understand the selection pressures on CYP450 genes among six cereal crop aphid species, we further calculated the ratios of the non-synonymous substitution rate (Ka) vs. the synonymous substitution rate (Ks). The average Ka/Ks ratios of duplicated CYP450 gene pairs across the whole genome of S. avenae, R. maidis, D. noxia, R. padi, S. graminum, and S. miscanthi were 0.85, 0.83, 0.40, 0.70, 0.81, and 0.62, respectively (Table S3). In addition, the Ka/Ks ratios of 61, 18, 13, 8, 5, and 2 duplicated CYP450 genes pairs across the whole genome of S. avenae, R. maidis, D. noxia, R. padi, S. graminum, and S. miscanthi were less than 1, indicating that purifying selection has been applied to these CYP450 genes during their evolutionary history. However, the Ka/Ks ratios of the remaining 12, 6, 1, 2, and 2 duplicated CYP450 genes pairs across the whole genome of S. avenae, R. maidis, R. padi, D. noxia, and S. graminum were larger than 1, indicating that these CYP450 genes have experienced a strong positive selection.

2.6. Transcriptomic Analysis of Rhopalosiphum Padi at Different Developmental Stages

We further investigated the expression profiles of R. padi from mature embryo to first instar through RNA data. A total of 26,301 genes were identified, of which 6611 genes had no expression at both developmental stages (Table S5). Differential expression analysis found that there were 1877 differently expressed genes between mature embryo and first instar, of which 680 DEGs were up-regulated expressed genes and 1197 DEGs were down-regulated expressed genes (Figure 6A–C). When aphid eggs of R. padi developed from mature embryo to first instar, down-regulated expressed genes played a major regulatory role. GO enrichment analysis found that all DEGs were enriched into three functional classifications (Figure 6D). In the molecular function category, the primary enrichment of DEGs was oxidoreductase activity, structural molecule activity, and structural constituent of cuticle catalytic activity. In the biological process category, DEGs were mainly enriched in small-molecule metabolic process, lipid metabolic process, and lipid transport. And in the cellular component category, DEGs were mainly enriched in the extracellular region, extracellular space, and cell periphery. KEGG enrichment analysis found that DEGs were mainly enriched in the pathways of fatty acid degradation, lipid metabolism, fat digestion and absorption, the glycosphingolipid biosynthesis-ganglio series, and cytochrome P450 (Figure 6E). Moreover, we found that 19 RpadCYPs were differently expressed between mature embryo and first instar, in which 15 RpadCYPs were up-regulated expressed and 4 RpadCYPs were down-regulated expressed, indicating that up-regulated RpadCYPs played a crucial role at the mature embryo stage of R. padi (Figure S9A,E).

2.7. Transcriptomic Analysis of Diuraphis Noxia Fed on Wheat Plants Containing Dn0, Dn4, or Dn7

We further investigated the expression profiles of D. noxia when fed on wheat plants containing the different resistance genes, Dn0, Dn4, or Dn7. The expression profiles and differential expression analysis were conducted among three groups (Dn4 vs. Dn0, Dn7 vs. Dn0, and Dn7 vs. Dn4). A total of 12,250 genes were identified, of which 1189, 1161, and 1168 genes had no expression among the Dn4 vs. Dn0, the Dn7 vs. Dn0, and the Dn7 vs. Dn4 groups, respectively (Table S6). Differential expression analysis found that there were 17, 36, and 15 differently expressed genes among the Dn4 vs. Dn0, the Dn7 vs. Dn0, and the Dn7 vs. Dn4 groups, respectively (Figure 7A). The Dn7 vs. Dn0 group had the most differently expressed gene number. In detail, there were 12 up-regulated expressed genes and 5 down-regulated expressed genes in the Dn4 vs. Dn0 group, and 21 were up-regulated expressed genes and 15 were down-regulated expressed genes in the Dn7 vs. Dn0 group, and 9 were up-regulated expressed genes and 6 were down-regulated expressed genes in the Dn7 vs. Dn4 group. It was up-regulated expressed genes that played a major role in all groups. Furthermore, three groups had no unique gene set. There were four genes (Dnox007922.1, Dnox001200.1, Dnox012221.1, and Dnox001645.1) existing in both the Dn4 vs. Dn0 and Dn7 vs. Dn0 groups. There was only one gene (Dnox002046.1) existing in both the Dn4 vs. Dn0 and the Dn7 vs. Dn4 groups. And there were five genes (Dnox005282.1, Dnox003280.1, Dnox011908.1, Dnox006834.1, and Dnox005642.1) existing in both the Dn7 vs. Dn0 and Dn7 vs. Dn4 groups (Figure 7B). The expression patterns of DEGs among these three groups were differential (Figure 7C–E). Due to fewer DEGs among the three groups, the union of DEGs among the three groups was used for the GO and KEGG enrichment analysis (Figure 7F,G). Results found that these genes were enriched in the following go terms: extracellular region, extracellular space, transporter activity, transmembrane transporter activity, and regulation of biological quality. KEGG pathways, including peptidases and inhibitors, metabolism, immune system, and signaling and cellular processes, were significantly enriched. Moreover, we found that there were no differently expressed DnoxCYPs in both the Dn4 vs. Dn0 and Dn7 vs. Dn4 groups, and only one gene DnoxCYP19 (Dnox005792.1) was differently up-regulated expressed in the Dn7 vs. Dn0 group, indicating that DnoxCYP19 played a crucial regulatory role when D. noxia interacted with the resistance gene Dn7 (Figure S9B,F).

2.8. Transcriptomic Analysis of Schizaphis Graminum Fed on Wheat Plants Dn0, Dn4, or Dn7

Similarly, we investigated the expression profiles of S. graminum when fed on wheat plants containing the different resistance genes Dn0, Dn4, or Dn7. The expression profiles and differential expression analysis were conducted with the Dn4 vs. Dn0 group, the Dn7 vs. Dn0 group, and the Dn7 vs. Dn4 group. A total of 26,072 genes were identified, of which 10,375, 10,342, and 10,249 genes had no expression among the Dn4 vs. Dn0 group, the Dn7 vs. Dn0 group, and the Dn7 vs. Dn4 group, respectively (Table S7). Differential expression analysis found that there were 16, 111, and 189 differently expressed genes among the Dn4 vs. Dn0 group, the Dn7 vs. Dn0 group, and the Dn7 vs. Dn4 group, respectively (Figure 8A and Figure S10A–C). The Dn7 vs. Dn4 group had the most differently expressed gene number. In detail, there were ten up-regulated expressed genes and six down-regulated expressed genes in the Dn4 vs. Dn0 group, indicating that the up-regulated expressed genes played a major role when S. graminum resisted the resistance gene Dn4. And there were 37 up-regulated expressed genes and 74 down-regulated expressed genes in the Dn7 vs. Dn0 group, and 56 up-regulated expressed genes and 133 down-regulated expressed genes in the Dn7 vs. Dn4 group, implying that down-regulated expressed genes played a critical role when S. graminum resisted with the resistance gene Dn7. Furthermore, three groups had only one unique gene (Sgrm022772.1). There were four genes (Sgrm007538.1, Sgrm011949.1, Sgrm024309.1, and Sgrm022772.1) existing in both the Dn4 vs. Dn0 and Dn7 vs. Dn0 groups. There were three genes (Sgrm015683.1, Sgrm012998.1, and Sgrm022772.1) existing in both the Dn4 vs. Dn0 and Dn7 vs. Dn4 groups. And there were 61 genes existing in both the Dn7 vs. Dn0 and Dn7 vs. Dn4 groups (Figure 8B). The expression patterns of DEGs among these three groups were differential (Figure 8C–E). Due to fewer DEGs in the Dn4 vs. Dn0 group, GO and KEGG enrichment analysis had no result. Results of GO enrichment found that the DEGs from the Dn7 vs. Dn0 group were enriched in the following go terms: hydrolase activity, glycosyltransferase activity, catalytic activity (molecular function), extracellular region (cellular component), organic acid metabolic process, organic acid biosynthetic process, and small molecule biosynthetic process (biological process) (Figure S10D). KEGG pathways of the Dn7 vs. Dn0 group, including organismal systems, lipid metabolism, peptidases, and inhibitors were significantly enriched (Figure S10F). The results of GO enrichment found that the DEGs of the Dn7 vs. Dn4 group were enriched in the go terms: hydrolase activity, catalytic activity, glucosyltransferase activity (molecular function), cell periphery (cellular component), organic acid metabolic process, organic acid biosynthetic process, and aspartate family amino acid metabolic process (biological process) (Figure S10E). KEGG pathways of the Dn7 vs. Dn4 group, including organismal systems, the digestive system, and metabolism, were significantly enriched (Figure S10G). Moreover, we found that there were no differently expressed SgrmCYPs in the Dn7 vs. Dn0 group. Only one gene, SgrmCYP16 (Sgrm006932.1), was differently expressed in the Dn4 vs. Dn0 group, indicating that SgrmCYP16 played a crucial regulatory role when D. noxia interacted with the resistance gene Dn4 (Figure S9C,G). SgrmCYP26 (Sgrm012776.1), SgrmCYP29 (Sgrm013967.1), and SgrmCYP47 (Sgrm023475.1) were down-differently expressed in the Dn4 vs. Dn7 group (Figure S9D,H), indicating that these CYP450 genes might interact with the resistance gene Dn4.

2.9. Transcriptomic Analysis of Winged and Unwinged Sitobion Avenae Aphids

We further investigated the expression profiles of S. avenae, including different tissues (body, head, whole organism) of unwinged aphids and the whole organism of winged aphids. The expression profiles and differential expression analysis of unwinged aphids were conducted among three groups (body vs. head group, whole organism vs. body group and whole organism vs. head group). A total of 31,007 genes were identified, of which 5857, 5312, and 5778 genes had no expression among the body vs. head group, whole organism vs. body group, and whole organism vs. head group, respectively (Table S8). Differential expression analysis found that there were 2865, 287, and 2114 differently expressed genes among the body vs. head group, whole organism vs. body group, and whole organism vs. head group, respectively (Figure 9A). The body vs. head group had the most differently expressed gene number. In detail, there were 1272 up-regulated expressed genes and 1593 down-regulated expressed genes in the body vs. head group, 252 were up-regulated expressed genes and 35 were down-regulated expressed genes in the whole organism vs. body group, and 1091 were up-regulated expressed genes and 1023 were down-regulated expressed genes in the whole organism vs. head group. We found that down-regulated expressed genes were larger than up-regulated expressed genes in the body vs. head group. However, up-regulated expressed genes were larger than down-regulated expressed genes in the whole organism vs. body and whole organism vs. head groups, indicating that up-regulated expressed genes were critical for whole organism tissue. Furthermore, these three groups had a unique gene set with 196 DEGs (Figure 9B). There were 1914 DEGs existing in both the body vs. head group and the whole organism vs. head group. There were 207 DEGs existing in both the whole organism vs. body group and the whole organism vs. head group. And there were 244 DEGs existing in both te body vs. head group and the whole organism vs. body group. The expression patterns of DEGs among different tissue groups were differential (Figure 9C–E). The intersection of DEGs among these three groups was used for the GO and KEGG enrichment analysis. Results found that DEGs were enriched in the go terms: lipase activity, passive transmembrane transporter activity, channel activity (molecular function), rhabdomere, extracellular space, obsolete extracellular region part (cellular component), phototransduction, detection of light stimulus, and rhodopsin-mediated signaling pathway (biological process) (Figure 9F). KEGG pathways, including sensory system, ion channels, amino acid metabolism, signaling, and cellular processes were significantly enriched (Figure 9G). Moreover, we found that there were 22, 2, and 11 differently expressed SaveCYPs in the body vs. head group, whole organism vs. body group, and whole organism vs. head group, respectively. These differently expressed SaveCYPs might play a significant role across the tissue development of S. avenae (Figure S11A–C,E–G).

Additionally, the whole organism of unwinged and winged S. avenae aphids were used to analyze the expression profiles and differential expression analysis. A total of 31,007 genes were identified, of which 5380 genes had no expression (Table S8). Differential expression analysis found that there were 233 differently expressed genes, of which 10 DEGs were up-regulated expressed genes and 223 DEGs were down-regulated expressed genes (Figure S12A,B). It is obvious that down-regulated expressed genes were more prevalent than up-regulated expressed genes, indicating that these down-regulated DEGs might negatively regulate the wing growth and development of S. avenae (Figure S12C). The results of GO and KEGG enrichment analysis found that these DEGs were enriched in the go terms: actin filament binding, transmembrane signaling receptor activity, structural molecule activity, signaling receptor activity (molecular function), external encapsulating structure, extracellular matrix, obsolete extracellular region part, extracellular region (cellular component), positive regulation of organelle assembly, regulation of actin filament polymerization, actin filament polymerization, regulation of actin polymerization or depolymerization, and regulation of actin filament length (biological process). KEGG pathways, including cardiac muscle contraction, exosome, enzymes with EC numbers, and lipid metabolism were significantly enriched (Figure S12D,E). Moreover, we found that there was only one differently expressed gene (SaveCYP39) which was differently up-regulated expressed in the unwinged whole organism vs. winged whole organism group, indicating that SaveCYP39 played a crucial regulatory role in the wing development of S. avenae (Figure S11D,H).

2.10. Transcriptomic Analysis of Sitobion Avenae Aphids Fed on Wheat Treated with Imidacloprid

In order to investigate the metabolic detoxification function of CYP450 genes in aphids towards insecticides, the RNA-seq analysis of S. avenae aphids fed on wheat treated with imidacloprid was conducted. Results showed that a total of 31,007 genes were identified, of which 6213 genes had no expression among imidacloprid treatment vs. control (Table S9). Differential expression analysis found that there were 119 differently expressed genes, of which 60 DEGs were up-regulated expressed genes and 59 DEGs were down-regulated expressed genes (Figure 10A,B). The expression patterns of DEGs when S. avenae aphids fed on wheat treated with imidacloprid are shown (Figure 10C). The results of GO enrichment analysis found that these DEGs were enriched in the go terms: unfolded protein binding, DNA-binding transcription factor activity (RNA polymerase II-specific), DNA-binding transcription factor activity (molecular function), extracellular space, extracellular region (cellular component), protein folding, response to starvation, response to extracellular stimulus, multicellular organism aging, obsolete cofactor metabolic process, and response to external stimulus (biological process) (Figure 10D). KEGG pathways, including chaperones and folding catalysts, antigen processing and presentation, protein processing in endoplasmic reticulum, and protein families’ metabolism were significantly enriched (Figure 10E). Moreover, we found that there was only one differently expressed gene SaveCYP48 (g21172.t1) was differently up-regulated expressed S. avenae aphids fed on wheat treated with imidacloprid, indicating that SaveCYP48 played a crucial regulatory role when S. avenae aphids resisted the toxicity to imidacloprid (Figure S13).

3. Discussion

Cytochrome P450 monooxygenases (P450s) are a large gene superfamily of heme thiolate proteins in prokaryotes or eukaryotes [36]. In addition to detoxifying exogenous toxic compounds like insecticides and plant secondary insecticides, cytochrome P450s play a role in the synthesis and degradation of endogenous compounds in insects, including hormones and sex pheromones. They also mediate insect growth and development, host plant adaptation, and insecticide resistance [37]. To date, the CYP450 gene family has been identified and characterized in prokaryote (cyanobacteria) [38] and fungi [39]. And the CYP450 gene family has also been analyzed in various plants species, such as rice [40], tomato [41], wheat [42], maize [42], sorghum [43], soybean [44], tea [45], and others. In insects, it was reported that the CYP450 gene family of Drosophila melanogaster [46], silkworm (Bombyx mori) [47], Plutella xylostella [48], Scopula subpunctaria [49], cotton leafhopper (Amrasca biguttula) [50], bees [51], and Bemisia tabaci [37] have been identified and analyzed. However, several aphid species had not been reported yet. In this study, we identified a total of 1100 CYP450 genes in 19 aphid species at the genome level. The CYP450 gene family of A. pisum, M. persicae, A. Gossypii, and A. Glycines owned 83,115,49 genes, and 68 members have been reported [52], while 66, 60, 54, and 60 CYP450s were identified in our results, which may be due to the strict parameters for identifying gene families. Furthermore, the 300 CYP450 genes belonged six cereal crop aphids, including S. avenae, S. graminam, S. miscanthi, R. padi, R. maidis, and D. noxia, which we focused on. It is generally believed that the insect CYP450 gene family consists of four branch clusters, namely the CYP2 clan, CYP3 clan, CYP4 clan, and the Mito clan [20]. According to the phylogenetic relationships and conserved motifs among six cereal crop aphids, these 300 CYP450 genes were classified into four groups, which we named CYP I, CYP II, CYP III, and CYP IV. CYP I belonged to the CYP3 clan, CYP II belonged to the CYP4 clan, CYP III belonged to the Mito clan, and CYP IV belonged to the CYP2 clan. The classification was consistent with that of the above reported insects.

In insects, cytochrome P450s have a role in their growth and development. Previous research revealed that, in comparison to other developmental phases, the expression of the TcCYP6BQ8 gene was comparatively higher in the early and late-larval stages of Tribolium castaneum [53]. In our study, we analyzed the expression profiles of R. padi from the mature embryo stage to the first instar stage, and found that 15 RpadCYPs were highly expressed at the mature embryo stage, suggesting that CYP450 genes mainly participated in the growth and development of mature embryos. According to tissue expression profiles of Tribolium castaneum, TcCYP6BQ8 is mostly expressed in the head and integument of both larvae and adults [53]. In another research, Pocyp4d2 had the highest expression in the midgut of Phortica okadai [54]. It was reported that SsubCYP341A and SsubCYP341B_ortholog1 were more highly expressed in the pheromone gland than in the female body of Scopula subpunctaria [49]. In our study, we obtained and analyzed the expression profiles of the unwinged S. avenae aphids among different tissues (body, head, and the whole organism). And we found that most of the SaveCYPs group had a higher expression level in the head than in the body, indicating that the CYP450 genes preferred to be expressed in the head tissue of S. avenae. In addition, we found that SaveCYP39 was down-regulated in the winged Sitobion avenae aphids, and thus SaveCYP39 might negatively regulate the wing growth and development of S. avenae.

The primary detoxifying enzyme system in insects is cytochrome P450, which is involved in the metabolism of a variety of pesticides, as well as other exogenous and endogenous substances [21]. Increasing CYP450 enzyme activity and overexpressing CYP450 genes were linked to pesticide resistance in a variety of insects [55]. Previous research revealed that the diamondback moth population resistant to chloramphenicol expressed much more of the P450 gene CYP6BG1 than susceptible populations [56]. When Tribolium castaneum was exposed to turpentene-4-ol, the expression of TcCYP6BQ8 was significantly induced. And silencing the cytochrome P450 gene TcCYP6BQ8 increased the larval mortality rate induced by turpentene-4-ol from 47.78% to 66.67% [53]. In addition, cotton specific aphids and cucumber specific aphids fed on epigallocatechin gallate (EGCG) and cucurbitacin B (CucB) significantly induced the expression of AgoCYP6CY19 [57]. When fourth-instar larvae were subjected to LC₅₀ dosage levels of indoxacarb, the death rate rose due to RNA interference-mediated CYP6AE68 silencing [55]. In our study, we analyzed transcriptome of D. noxia and S. graminum fed on wheat plants containing different resistance genes, Dn4 or Dn7, compared to Dn0. DnoxCYP19 (Dnox005792.1) was differently up-regulated expressed when D. noxia fed on wheat plants containing different resistance genes, Dn7, indicating that DnoxCYP19 might participate in weakening the resistance gene Dn7. However, SgrmCYP16 (Sgrm006932.1) was differently up-regulated expressed when S. graminum fed on wheat plants containing different resistance genes, Dn4, indicating that DnoxCYP19 might participate in weakening the resistance gene, Dn4. To explore whether the CYP450 gene family was related to the detoxification of insecticides in aphids, we also analyzed the transcriptome of S. avenae aphids fed on wheat treated with imidacloprid. Finally, we found that SaveCYP48 (g21172.t1) was differently up-regulated expressed in S. avenae aphids fed on wheat treated with imidacloprid, indicating that SaveCYP48 might play a crucial regulatory role when S. avenae aphids resisted the toxicity to imidacloprid. In conclusion, our study systematically identified and analyzed CYP450 genes in 19 aphid species, providing some useful clues for the further molecular and functional identification of the CYP450 gene family. The transcriptome profiles of aphids at different developmental stages, different tissues, and when fed on wheat plant containing resistance genes or imidacloprid provide new potential targets for aphid control and management.

4. Materials and Methods

4.1. Genome-Wide Identification of CYP450 Genes in 19 Aphid Species

The genome, CDS, protein, and genome annotation file of 18 aphid species, except S. avenae, were obtained from the InsectBase 2.0 (http://v2.insect-genome.com/ (accessed on 15 November 2022)) as the local database. The genome, CDS, protein, and genome annotation file of S. avenae were obtained from the figshare website (https://figshare.com/ (accessed on 13 November 2022)) [58] (Table S10). Then, using the HMMER 3.0 tool with e-value < 1 × 10⁻¹⁰, the CYP450 typical domain (PF00067) was retrieved from the PFAM database (http://pfam-legacy.xfam.org/ (accessed on 16 November 2022)) and used as the query to search against the protein database of every species of aphid. The protein sequences identified with above method were regarded as candidates of CYP450 gene family. To confirm the existence of the Cytochrome P450 superfamily domain, the candidates were then submitted to the NCBI-CDD (https://www.ncbi.nlm.nih.gov/cdd/ (accessed on 2 December 2022)) and InterPro websites (http://www.ebi.ac.uk/interpro/ (accessed on 2 December 2022)). The proteins harboring the complete Cytochrome P450 superfamily domain were considered to be CYP450 genes. The putative CYP450 proteins were uploaded to the ExPASy database (https://web.expasy.org/protparam/ (accessed on 2 December 2022)) in order to calculate the grand average of hydropathicity (GRAVY), molecular weight (MW), and theoretical isoelectric point (pI). The WoLF PSORT program was used to predict the subcellular localization of them.

4.2. Phylogenetic Relationship, Gene Structure and Conserved Motif Analysis of CYP450 Genes among Six Cereal Crop Aphid Species

To understand the evolutionary relationship of CYP450 genes among cereal crop aphids, including D. noxia, R. maidis, R. padi, S. graminum, S. miscanthi, and S. avenae, multiple sequence alignments of the discovered CPY450 proteins of these cereal crop aphids were carried out using the ClustalW v2.0 program [59]. The phylogenetic tree was created using the MEGA-X program using the neighbor-joining approach with a 1000 replication bootstrap [60]. The tree of phylogenetic relationships was improved using EVOLVIEW (https://evolgenius.info//evolview-v2/#login (accessed on 7 December 2022)). The chromosomal location and exon-intron structures of CYP450 genes were obtained from genome annotation files (gff3). MapGene2Chromosome v2.0 (http://mg2c.iask.in/mg2c_v2.0/ (accessed on 16 January 2023)) was used to display the physical positions of the CYP450 genes on the chromosomes, and Gene Structure Display Server (GSDS2.0) (https://gsds.gao-lab.org/ (accessed on 8 December 2022)) was used to display the exon-intron structures. The MEME v5.2.0 program was used to find the conserved motifs of CYP450 proteins, with a maximum of ten motifs allowed [61].

4.3. Gene Duplication and Synteny Analysis of CYP450 Genes among Six Cereal Crop Aphid Species

To understand expansion events of the CYP450 gene family, including gene duplication and tandem duplication, MCScanX software was utilized to evaluate syntenic relationships among all CYP450 genes present in cereal crop aphids. The Circos tool was used to display the linked gene pairs. The software MCScan (Python-jcvi) was utilized to identify syntenic links between the various genomes of cereal crop aphids. Using the KaKs_Calclator 2.0 program, the synonymous (Ks) and nonsynonymous (Ka) substitution rates were determined.

4.4. RNA-seq Data Analysis of Four Cereal Crop Aphid Species

To get additional insight into the expression patterns and spatial-temporal expression of these CYP450 genes among cereal crop aphids, we collected RNA-seq data of four cereal crop aphids, including R. padi, D. noxia, S. graminum, and S. avenae, which were obtained from Sequence Read Archive database (http://www.ncbi.nlm.nih.gov/sra (accessed on 9 February 2023)) (Table S11). The wheat plants harboring distinct resistance genes Dn0, Dn4, or Dn7 were fed to the aphid biotypes of S. graminum and D. noxia, identified as S. graminum biotype I and D. noxia biotype 1, respectively. The transcriptomes of S. graminum biotype I and D. noxia biotype 1 were compared using the wheat varieties Yuma, which contains no resistance genes (Dn0), Yumar, which contains a Dn4 resistance gene [62], and 94M370, which contains a Dn7 gene for resistance to D. noxia biotype 2 [63].

The RNA data of R. padi were at the stages of the mature embryo and first instar. And the RNA data of S. avenae contained the body, head, and whole organism of unwinged and winged aphids. Moreover, we collected the RNA-seq data of S. avenae aphids fed with wheat treated with imidacloprid. Hisat2 was used to map this RNA-seq data to the appropriate local reference genome. StringTie v2.1.2 was used to determine the FPKM of each gene (fragments per kilobase of transcript per million fragments of mapped reads) value [64]. The featureCounts program was used to compute raw counts.

4.5. Differential Expression Analysis among Four Cereal Crop Aphids

DESeq was used to filter the differentially expressed genes (DEGs) among the several comparison groups according to the following standards: |log2 FC| ≥ 1, and p value < 0.05. Using the Z-score approach, the FPKM of all DEGs across various comparison groups were normalized. The pheatmap package and ggplot2 package in R software (4.0.3) were used to create a heatmap and a volcano plot to visualize the expression patterns.

4.6. GO Enrichment and KEGG Pathway Enrichment of DEGs among Four Cereal Crop Aphids

The DEGs were subjected to GO enrichment and KEGG pathway enrichment analysis by utilizing the TBtools program [65]. A BackGround file (Query2Go and Query2Knum) was extracted from the gene annotation file. The bubble charts of GO enrichment and KEGG enrichment were visualized using the clusterProfiler package.

4.7. Expression Patterns of Differential Expressed CYP450 Genes among Four Cereal Crop Aphids

In order to comprehend the patterns of spatial-temporal expression of CYP450 genes among cereal crop aphids, the differential expressed CYP450 genes between the above different comparison groups were extracted to further display the expression profiles of CYP450 family genes. Through the Z-score approach, the FPKM of CYP450 family genes was normalized across several comparison groups. The pheatmap package and ggplot2 package in R software (4.0.3) were used to create a heatmap, and a volcano plot was used to visualize the expression patterns.

5. Conclusions

This is the first study to identify the cytochrome P450 gene family in 19 aphid species at the genome level. A total of 1100 CYP450 genes were identified in 19 aphid species, and 300 CYP450 genes were belonged to 6 cereal crop aphid species. The members of S. flava had the largest average value of the gene length, CDS length, protein size, and molecular weight. However, the members of A. solani had the lowest average value of the gene length, CDS length, protein size, and molecular weight. Subcellular localization predictions revealed that most of CYP450 proteins were located in the endoplasmic reticulum, indicating that they mainly played a role in the endoplasmic reticulum. The predicted GRAVY value of all these CYP450 proteins were minus, indicating they attract water molecules or easily dissolve in water. According to phylogenetic relationship, these 300 CYP450 genes were further divided into four subfamilies (CYPI, CYPII, CYPIII, and CYPIV). The genomic organizations, exon-intron structures, and conserved motifs of the same subfamilies were similar. Almost all members of CYP I subfamily had all of the 10 motifs, the members of CYP II subfamily lacked motif 5 and motif 8, the members of CYP III subfamily lacked motif 5, and the members of CYP IV subfamily lacked motif 4 and motif 7, indicating that there are different motif organizations among these four subfamilies. The CYP450 members of R. maidis, R. padi, and S. graminum always clustered together on the same clade, indicating that R. maidis, R. padi, and S. graminum had a closer genetic relationship. And the CYP450 members of D. noxia, S. miscanthi, and the S. avenae always clustered together on the same clade, indicating that D. noxia, S. miscanthi, and S. avenae had a closer genetic relationship. The chromosome localizations and distribution of CYP450 genes among six cereal crop aphid species were differential. The gene expansion events of CYP450s in R. maidis and S. avenae possessed both gene duplications and tandem duplications, while only tandem duplications occurred in D. noxia, R. padi, S. graminum, and S. miscanthi. Moreover, three continuous colinear gene pairs among six cereal crop aphid species were found, which were very conserved among these species. In addition, most of duplicated CYP450 genes pairs across the whole genome of S. avenae, R. maidis, D. noxia, R. padi, S. graminum, and S.miscanthi underwent purifying selection during evolution. Furthermore, we systematically investigated the expression profiles of four cereal crop aphids, including R. padi, D. noxia, S. graminum, and S. avenae. Differential expression analysis provided growth stage specificity genes, tissue specificity genes, organ specificity genes, and some detoxification metabolic genes among these four cereal crop aphids. The differentially expressed CYP450 genes from all the DEGs were obtained, which provided some useful clues for further study to the functions of the CYP450 gene family, and provided some potential candidates for aphid management and control.