4. Discussion
The study of the multiple-omics techniques of polyphagous C. punctiferalisand oligophagous C. pinicolalis can initially, from a particular aspect reveal the reasons of the different dietary habits in the two species. In this study, the genes related to carbohydrate metabolism were selected and some of those genes were different in highly expression and sequence mutations. Actually, the differential expression or mutation of these genes is an important reason for their different eating habits.
A role for gene duplication or amplification in resistance or detoxicate has now been demonstrated for some species, both as a route of enhanced production of metabolic enzymes (Devonshire et al., 1991; Bass and Field, 2011). The increased production of metabolic enzymes, which can break down or bind to (sequester) the pesticide (Bass and Field, 2011). However, in this study, the differential expression of the two genes in different species is more closely related to their food. α-amylase is an oligosaccharide endoglycosidase, an enzyme that cleaves an internal glycosidic bond within a poly or oligosaccharide helps digest carbohydrates (starch and glycogen) into simple sugar [glucose (monosaccharide) and maltose (disaccharide)] for energy. α-amylase is highly expressed in C. punctiferalis may relationship with the kind of hosts. C. punctiferalis larvae have been reported to attack more than 100 essential plant species, including peach, durian, chestnut, citrus, papaya, cardamom, ginger, etc (Lu et al., 2010), and those plant can support more carbohydrates. On the contrary, the host ofC. pinicolalis is only Masson pine. The literature shows that Masson pine needles contain a lot of cellulose, fat and protein, etc (Tian, 2006; Nie et al., 2020). Therefore, the type of food is relatively simple, and the nutrients in it are limited, and the demand for amylase will be relatively low.
The genomes of phytophagous insects usually contain large numbers of P450s, especially within the CYP3 clan. Within this clan, CYP6 subfamily members help detoxify plant host secondary metabolites (Feyereisen, 2012; Mittapelly et al., 2019; Tzou et al., 2000). Knockout of the CYP6AE cluster does not affect the viability of the insect, but it results in increased susceptibility to both plant toxins and synthetic insecticides (Wang et al., 2018). In this study, C. punctiferalisas a polyphagous insect, CYP6AE76 gene is not only highly expressed in larvae (Figure 3), but the enzyme activity level is also significantly higher than oligophagous C. pinicolalis (Figure 4). Moreover, Mittapelly et al . (Mittapelly et al., 2019) reported that the CYP6 gene expression in polyphagous insects is not based on host diet, however, they might use a cocktail of broad-spectrum detoxification enzymes that interact with a variety of compounds encountered in their diets and these CYP6Bs may be part of that cocktail. Those results showed that polyphagous C. punctiferalis needs more CYP6AE76 to metabolize or detoxify substances from a variety of foods. On the contrary, oligophagous C. pinicolalis only eats pine needles, so there is no need for multiple detoxification and metabolism compared toC. punctiferalis . In addition, previous studies showing induction of some CYP6AE genes by specific chemicals or different host plants (Zhou et al., 2010; Celorio-Mancera et al., 2011). However, the pine needles may contain a small amount of specific chemical substances mentioned above, and the food source of C. pinicolalis is relatively single, so the expression of the CYP6AE76 gene will be relatively low.
Environmental conditions are not always suitable for survival, and insects employ multiple strategies for adaptation (Zhai et al., 2019). After long-term evolution, the two species of C. punctiferalis and C. pinicolalis have become more distinct in adults and can be distinguished by the mitochondrial cytochrome c oxidase gene (Wang et al., 2014; Jeong et al., 2021). In this study, after a multi-omics joint analysis, α-amylase and CYP6AE76 were found to have mutations. However, no mutations were detected in the homologous conserved regions and enzyme active sites in α-amylase (Figure 2A), and the sequence similarity could reach 94% (Table 3). On the contrary, mutations appeared in some other regions. Although these mutations do not cause structural changes, they may also differ significantly in their exact substrate preference and product profile (Janeček and Gabriško, 2016). Therefore, those mutations may have caused the high expression of α-amylase and its enzyme activity in C. pinicolalis , thus affecting their metabolism or detoxification of food. Although the P450 super-family has a wildly divergent sequence and the overall homology may be less than 40% even within the same family, particularly in insects (Wang et al., 1995), there are function-critical sequence motifs preserved during evolution heme-binding sequence motif (FxxGxxxCxG) universal among CYP enzymes. In this study, no mutations in the heme-binding site were detected which suggested the main function has not changed. However, all SRS sties of the two species have mutations. Some studies have demonstrated that amino acids in SRSs affect the protein folding and substrate range of cytochrome P450s, especially SRS1 in a loop region, close to the active site heme, has proven to be the most crucial SRS that affects multiple properties of P450s (Domanski and Halpert, 2001; Schuler and Berenbaum, 2003; Shi et al., 2020). Recently, Zuo et al (Zuo et al., 2021) revealed that the mutation located in the SRS1 region of CYP9A186 ofSpodoptera exigua , causes resistance to both emamectin benzoate and abamectin. In addition, target-site resistance involves alterations (e.g., mutations) in the insecticide target protein that reduce its sensitivity to insecticides (Zimmer et al., 2016). Therefore, due to these mutations, the binding ability is different, which indirectly leads to the different detoxification ability, and it has been verified by the qPCR and enzyme activity test. However, the difference of feeding habits of insects is result of their long-term adaptation to the environment, and genetic mutation is one of many factors. Therefore, further research is needed.
Metabolites are the end products of cellular regulatory processes. Therefore, it is necessary to understanding the final metabolites difference in dietary habits between the two species. The detection results showed more metabolic difference substances in C. punctiferalis than C. pinicolalis . Among the top 20 down-and up-accumulated metabolites, C. punctiferalis is mainly metabolizes of amino acids, organic acids, and alcohols; while C. pinicolalis mainly metabolizes of lipids, organic acids, and terpenes (Figure 5B). These differences can also reflect their different foods resource, especially in C. pinicolalis . The pine needles contain many lipids (Tian et al., 2006), so these enzymes are needed for metabolism, this may also be the reason why its α-amylase activity is stronger than that of C. punctiferalis . The KEGG classification indicates that C. punctiferalis is enriched in more metabolic pathways than C. punctiferalis . For example, biosynthesis of amino acids, pyrimidine metabolism, ATP binding cassette transporters (ABC) transporters, etc (Supplementary Figure 4). However, ABC as a transporter has been increasingly recognized with resistance to cancer chemotherapy in humans, drug resistance in protozoa, antibiotic resistance in bacteria, and pesticide detoxification in nematodes, arthropods and Lepidoptera pests in recent years (Lage, 2003; Porretta et al., 2016; Zhou et al., 2020). In the current study, although esterases, glutathione S-transferases and P450 as three prominent enzyme families are well known implicated in metabolic and digestive (Li et al., 2007), no significant differences were found in the metabolites of the two species. More interestingly, ABC transporters are higher in polyphagous C. punctiferalis than in oligophagous C. pinicolalis , suggesting polyphagous insects need more metabolism and transport pathways to degrade or digest different foods or toxins in foods. Therefore, more in-depth research is needed on ABC transporters.