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.