Evolution of gene families
Gene family expansion and contraction have been suggested as essential and fundamental adaptation mechanisms [50]. To reveal key gene family changes related to adaptation, gene family expansion and contraction in the P. micranthus genome were analyzed using CAFÉ by comparing with the genomes of A. lucorum, N. tenuis, A. gossypii, N. lugens, D. citri, and H. halys, A. glycines, R. maidis and C. lectularius.
After divergence from the ancestor of H. halys, N. lugens, D. citri, R. maidis, A. gossypii, and A.glycines, 450 and 4372 gene families were expanded and contracted in the P. micranthus genome (Fig. 2c). This finding suggested that many gene families in the P. micranthus genome were lost rather than gained during adaptive evolution.
The GO analysis revealed that the expansion genes were significantly enriched in various Go terms, such as transferase activity (GO:0016758), membrane (GO:0016020), proteolysis (GO:0006508), odorant binding (GO:0005549), cysteine-type peptidase activity (GO:0008234) as well as sensory perception of taste (GO:0050909) (Fig. 2d). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that most of the expanded genes were significantly enriched in carbohydrate metabolism, biosynthesis of secondary metabolites, metabolism of cofactors and vitamins, xenobiotics biodegradation and metabolism, lipid metabolism, signal transduction, cell growth and death, metabolism of terpenoids and polyketides and immune system (Fig. 2e). Significant expansion or contraction of gene families is commonly associated with the adaptive evolution of species [51, 52]. P. micranthus absorbs nutrients required to support its development and growth from M. micranthus leaves, and its oviposition also relays on M. micranthus stems. Thus, several significantly expanded gene families associated with chemoreceptor, digestion, and detoxification were considered necessary for adaptive evolution. These include the chemoreceptor annotations, such as odorant binding and sensory perception of taste; the digestion annotations, such as proteolysis and polygalacturonase activity; the detoxification annotations, such as drug metabolism-cytochrome P450 and metabolism of xenobiotics by cytochrome P450 (Additional file 2: Tables S17 and S18).
In addition, 11 positively selected genes were identified in P. micranthus, and then KEGG and GO analyses were done (Additional file 2: Tables S19 and S20). These genes involved some terms, such as lipid metabolism, genetic information processing, and signal transduction.
Analysis of gene families associated with P. micranthus adaptation to M. micrantha
Insect feeding behaviour is a complex process associated with initial activation, orientation, identification, and feeding [53]. As stated, the chemosensory system is crucial in locating food, mates, and oviposition [7, 8]. P. micranthus is an oligophagous insect, and myrcene has a robust and attractive effect on this bug [11]. It has been shown that olfactory cues could explain the physiological mechanism underlying host recognition [6, 54]. P. micranthus feed by inserting piercing-sucking mouthpart into M. micrantha leaves, injecting slavery enzymes, and then aspirating liquefied materials. Therefore, this plant bug is a typical extraoral digestion, piercing-sucking, and “lacerate and flush feeding” insect [16]. The liquefied materials are further digested and absorbed in the gut. As we stated previously, the digestive enzymes remaining in plants cause continuous tissue damage for an extended period, leading to a decrease in the growth rate and loss of flowers [18–20]. The digestive enzyme is an essential factor in the adaption of P. micranthus to M. micrantha and also provides a new strategy to control M. micrantha. The adaption of insects to the plants they feed on partially depends on detoxification genes [55]. Thus, the detoxification genes can further support P. micranthus feeding and adaptation to M. micrantha.
We manually identified 59 gustatory receptors (GRs), 12 ionotropic receptors (IRs), 40 OBPs, and 92 ORs in the P. micranthus genome (Fig. 3a), which were closely related to the encoding of significantly expanded gene families. This relevance may also indicate the specific recognition and adaptation mechanism of M. micrantha. GRs, ORs, and IRs are thought to help detect odours and function as chemosensory receptor multi-gene families in insects [8]. The three chemosensory receptor gene families are mainly expressed in insect chemosensory sensilla that harbour olfactory sensory neurons (OSNs) [56]. GRs are expressed in gustatory receptor neurons, which encode seven transmembrane domains [57, 58]. GRs mainly respond to non-volatile compounds, including sugars, salts, gustatory pheromones, bitter compounds, and carbon dioxide [59–62]. The number of GRs was the highest in P. micranthus compared with the other three mirid bugs, and the number of GRs in phytophagous was more than carnivorous (Fig. 3b), consistent with the previous study [10]. However, the number of GRs identified differed due to different identification and screening methods. ORs were the first family of chemosensory receptors to be discovered in insect OSNs, and their function depends on the highly conserved odorant receptor co-receptor (Orco) gene. Orco can form Orco-ORx complexes with conventional olfactory receptors rather than odorant ligands to improve the efficiency of traditional olfactory receptor responses to odours [63, 64]. In our study, the P. micranthus genome contained 99 OR genes, including one Orco, which clustered in one branch with Orco of A. lucorum (Additional file 1: Fig. S8b). Moreover, like omnivorous A. lucorum and H. halys, non-omnivorous P. micranthus and N. lugens also had higher ORs numbers than other insects, indicating that there is a clear variance in numbers of ORs among different insects. IRs are a class of ionotropic glutamate receptors (iGluRs) and consist of two subfamilies: antennal IRs and divergent IRs [65, 66]. Unlike GRs and ORs, the primary receptor proteins for detecting odorants and tastants, IRs mainly detect chemo-, thermo-, and hygro-sensory stimuli [56, 62, 65]. There was little difference in the number of IRs among different species, which suggested that IRs are evolutionarily conserved (Fig. 3a). OBPs are a class of water-soluble proteins (approximately 150 amino acids) widely found in the olfactory mucosa of vertebrates and the sensilla fluid of insects [67, 68]. The first member of the OBP family was identified in the antennae of male Antheraea polyphemus (Lepidoptera: Saturniidae) [69]. OBPs are responsible for carrying odorant molecules to chemoreceptors located on sensory neurons, and OBPs may also be related to olfactory gene encoding and stimulus inactivation [68]. Based on their primary protein sequences and conserved cysteine number, OBPs have been classified into four subfamilies: classical OBPs, plus-C OBPs, minus-C OBPs, and atypical OBPs in Diptera or Lepidoptera [70, 71]. Only two subfamilies of classical and plus-C OBPs are present in Hemiptera; for example, 12 classical OBPs and two plus-C OBPs in Adelphocoris lineolatus, 20 classical OBPs and 12 plus-C OBPs in L. lineolaris, 24 classical OBPs and two plus-C OBPs in Corythucha ciliata, and 19 classical OBPs and three plus-C OBPs in Yemma signatus [72–75]. Our research identified 33 OBPs in the P. micranthus genome (Fig. 3a), including 17 classical OBPs and 16 plus-C OBPs (Additional file 1: Fig. S9, S10a). Moreover, phylogenetic analysis showed species-specific GR, OR, and OBP genes clustered in the same clades (Additional file 1: Fig. S8a, 8b, and 10a). The duplication of those chemosensory genes may be related to the ability of P. micranthus to specifically recognize M. micrantha [76, 77].
In total, 34 lipases, 122 serine proteases (SPs), 20 polygalacturonases (PGs), 27 cysteine proteases (CPs), and five alpha-amylases were identified in the P. micranthus genome (Fig. 3b). Of these, SPs, PGs, and CPs are three significantly expanded gene families in P. micranthus (Fig. 2d). SP is widespread, including all kingdoms of cellular life and many viruses [78]. SPs are essential digestive enzymes in insects' physiological and pathological functions, such as fibrinolysis, development, fertilization, digestion, and immune defence [78, 79]. The main digestion-related functions of SPs are the breakdown of proteins into free amino acids and the degradation of plant toxins [21, 80]. Due to its diverse functionalities, the number of SPs in each species was significantly higher than that of other genes (Fig. 3b). PG is a vital cell hydrolysis enzyme. It causes visible plant injury, which catalyzes the hydrolysis of α-1,4-glycosidic linkages in polygalacturonic (pectic) acid in mirids, weevils, and a few other insect species [81–83]. PG has been widely described in fungi, bacteria, nematodes, and plants [84]. In insects, PGs have been reported in many orders with piercing-sucking and chewing mouthparts, beetles (Coleoptera, mainly of the Phytophaga clade) are included and notably common in mirid bugs (Hemiptera) [85–87]. Mirid PGs can cause much larger lesions than superficial mechanical damage or feeding by other sap-sucking insects [84]. Moreover, studies have found that microinjection of Lygus PG can cause cotton flower abortion [19]. On the contrary, many plants produce polygalacturonase-inhibiting proteins (PGIPs) to reduce insect PG activity [88]. Among 11 Hemiptera insects, PGs were identified only in four mirid bugs: P. micranthus (20), A. lucorum (52), N. tenuis (6), and C. lividipennis (1) (Fig. 3b). The food sources that insects can obtain determine the type and quantity of digestive enzymes [89]. The number of PGs in the P. micranthus genome was lower than that of the omnivorous plant bug A. lucorum and higher than that of the carnivorous N. tenuis and C. lividipennis (Fig. 3b). Furthermore, the phylogenetic analysis showed that the PGs of each mirid bug were primarily clustered in one species-specific clade, suggesting that PGs were evolutionarily conserved (Additional file 1: Fig. S10b). CP is an essential group of proteolytic enzymes in insects and has been reported in Drosophila melanogaster, Tenebrio molitor, Tribolium castaneum, and Frankliniella occidentalis [90–93]. Because CPs show better activity and stability at a slightly acidic pH (5–7), they are mainly found in the anterior midgut [94, 95]. CPs are associated with the hydrolysis of yolk proteins, protein turnover in lysosomes, and tissue decomposition [96]. There was small difference for the number of CPs between these species (Fig. 3b), it may be in part due to the function of CPs were diverse. However, the number of CPs in oligophagous species was generally lower than that of omnivorous species. Therefore, the low number of CPs in oligophagous species may be due to its narrow host range. Plants, like PGIPs, can synthesis cysteine peptidase inhibitors to inhibit CP activity [97, 98]. Based on this, M. micrantha can be controlled by RNA interference (RNAi) to inhibit the synthesis of PGIPs and cysteine peptidase inhibitors or to develop environment-friendly specific biological control agents.
Lastly, we manually identified four important detoxification enzyme families in the P. micranthus genome, including 31 GSTs, 69 P450s, 56 CCEs, and 44 ABCs (Fig. 3c). P. micranthus had the highest number of CCE genes and the second highest number of GST genes and P450 genes compared to the other four mirid bugs. The number of ABCs genes in P. micranthus is the second lowest and slightly higher than N. tenuis compared to the other hemipteran insects. These findings illustrated that P. micranthus might have a unique way of metabolizing toxic substances from food and the environment. Among these four detoxification gene families, as we described before, P450 was a significantly expanded gene family in P. micranthus that was annotated in the KEGG enrichment analysis (Fig. 2e). Cytochrome P450, or CYP genes, is one of the most prominent gene families, broadly distributed in nearly all living organisms [99, 100]. P450s are involved in the synthesis and metabolism of endogenous compounds and the metabolism of many exogenous compounds, such as a series of pesticides, hormones, steroids, fatty acids, and plant toxins [101, 102]. Therefore, P450s are crucial for insects to adapt successfully to their host plants [103]. According to the evolutionary relationships in the phylogenetic tree, insect P450s are divided into four clades: CYP2, CYP3, CYP4, and mitochondrial (Mito) [104, 105]. In total, 6 CYP2, 36 CYP3, 22 CYP4, and 5 Mito genes were identified in the P. micranthus genome, and the number of CYP3 genes was the largest (Fig. 4a). Phylogenetic analysis showed that many P450 genes were grouped in the CYP3 and CYP4 clades. Mapping of P450 genes to P. micranthus chromosomes showed that the CYP3 and CPY4 genes were mainly mapped to 2, 3, 6, and 10 chromosomes; in particular, chromosome 10 was only clustered many CYP3 genes (Fig. 4b). These results indicated that CYP3 genes experienced a relatively recent species-specific expansion in P. micranthus. CYP3 is pivotal in detoxifying plant secondary metabolites and pesticide resistance [106]. The expansion of CYP3 genes in P. micranthus might be associated with its specific detoxification of toxic substances of M. micrantha and evolutionary adaptation to M. micrantha.
Salivary gland transcriptome analysis reveals a high feeding efficiency of P. micranthus
Following the assembly, 11,746 genes were generated using Histat2 in the salivary gland transcriptome, consistent with the result in genome annotation (Table 2). Gene enrichment analysis showed that among the 11,746 genes, 7814 genes were associated with 7102 GO terms, and 6364 genes were associated with 437 KEGG Orthology (KO) terms.
To further understand the gene expression in the salivary gland, the expression of the genes in the salivary gland was then compared to the expression of the genes in the whole body. Genes with an absolute fold change equal to or greater than 2.0 and a p-adjusted value (p.adj) less than or equal to 0.05 were considered differentially expressed. Using these criteria, we obtained 1593 downregulated genes, 7015 not differentially expressed, and 2798 upregulated genes (Fig. 4c). The upregulated genes were more specific and highly expressed in the salivary gland than in the whole body. KEGG and GO enrichment analysis of the upregulated genes were further performed using clusterProfiler based on Evolutionary genealogy of genes: Non-supervised Orthologous Groups (EggNOG) annotations. KEGG enrichment analysis revealed that most of the upregulated genes were significantly (p.adj ≤ 0.05) involved in metabolism pathways, such as Peptidases and inhibitors (ko01002, contained179 genes), Protein digestion and absorption (ko04974, contained 53 genes), Galactose metabolism (ko00052, contained 41 genes), Glutathione metabolism (ko00480, contained 41 genes), Cytochrome P450 (ko00199, contained 40 genes) (Fig. 4d). Of interest, glutathione plays a vital role in plant disease resistance, cell proliferation, root development, salt tolerance, and cold injury protection [107]. The pathways of “Glutathione metabolism” and “Cytochrome P450” in insects were beneficial for inhibiting plant defence response and metabolizing and detoxifying xenobiotics from the plant [103]. In addition, 19 genes were significantly enriched in “Salivary secretion” (ko04970), and some enriched KEGG pathways contained downregulated genes (Fig. 4d). For GO enrichment analysis, for easier visualization, only displayed the top ten GO terms for different aspects (biological process, cellular component, and molecular function), respectively (Fig. 4e). Notably, nearly all Go terms were associated with peptidase activity in molecular function, especially among cysteine peptidase, serine peptidase, and polygalacturonase (Fig. 4e). The expanded gene families of P. micranthus also significantly enriched most of these GO terms (Fig. 2d). Lygus linearis salivary gland genes were also significantly enriched in those terms [17], which revealed a similar enrichment pattern of the two mirid bugs. However, apart from Miridae, even phytophagous Hemipteran belonging to the same family showed different gene enrichment patterns in salivary gland transcriptome, such as Nephotettix cincticeps (Cicadellidae) [108], Nephotettix cincticeps (Cicadellidae) [109], Sogatella furcifera (Delphacidae) [110], and Bemisia tabaci (Aleyrodidae) [111]. Therefore, these results may illuminate that phytophagous mirid bugs has a specific salivary enzyme system. Since the study of mirid bugs’ salivary glands was rare, this needs to be explored further. Furthermore, the highly expressed genes in the P. micranthus salivary gland were significantly associated with metabolism pathway, peptidase activity, cysteine peptidase, serine peptidase, and polygalacturonase, which might also be a reason for precisely and highly efficiently feeding by P. micranthus on M. micrantha, and provides the availability of this oligophagous bug for controlling M. micrantha.