Abstract

   Plants are important ecological factors and food resources, which can
   significantly affect the occurrence and distribution of insects. The
   metabolites in host plants can affect the feeding, spawning, and
   avoidance behaviors of herbivorous insects. Galeruca daurica (Joannis)
   is a phytophagous pest that has seriously occurred in the desert steppe
   of Inner Mongolia in recent years, only infesting the leaves of Allium
   plants. In order to clarify the effects of plant metabolites on the
   gene expression in G. daurica larvae at the transcriptome level, we fed
   the larvae of G. daurica with Allium tuberosum leaves soaked in 10%
   DMSO solutions containing d-galactose, β-d-glucopyranose, l-rhamnose,
   isoquercitrin, isoflavone, and rutin, respectively, used the larvae fed
   on A. tuberosum leaves soaked in a 10% DMSO solution as the control,
   and screened out the differentially expressed genes (DEGs) by
   performing high-throughput transcriptome sequencing. The results showed
   that a total of 291 DEGs were identified compared to the solvent
   control (DMSO), including 130, 34, 29, 21, 72, and 97 in the
   isoquercitrin, isoflavone, rutin, d-galactose, β-d-glucopyranose, and
   l-rhamnose treatment groups, respectively. GO and KEGG enrichment
   analysis showed that most DEGs were enriched in various metabolic
   pathways, implying that these six main primary and secondary
   metabolites in Allium plants may affect various metabolic processes in
   the larvae of G. daurica.

1. Introduction

   In the long-term evolutionary process, there is a close and complex
   relationship between herbivorous insects and host plants. On the one
   hand, host plants provide a source of nutrients for herbivorous
   insects, and on the other hand, they also affect the biological
   characteristics of herbivorous insects, such as growth and reproduction
   [[34]1,[35]2]. Many factors affect insect feeding, among which
   phytochemical properties are the main ones, including plant nutrients
   or primary metabolites and secondary metabolites [[36]3,[37]4]. Primary
   metabolites include carbohydrates, lipids, nucleic acids, and proteins.
   For example, feeding on carbohydrates, such as sucrose, glucose, and
   fructose, is beneficial for insect survival and reproduction
   [[38]5,[39]6]. Plant secondary metabolites are usually divided into
   three broad classes: terpenoids, phenolics (polyphenols), and
   alkaloids, among which phenolics are the largest and most diverse and
   most widely distributed class, which mainly include flavonoids,
   phenolic acids, stilbenes, and lignans [[40]7]. Flavonoids, which also
   include flavones, flavanols, flavans, anthocyanins, and isoflavones,
   are secondary biomasses with predominantly anti-insect functions and
   have been shown to play an important role in the anti-insect process of
   plants [[41]8,[42]9]. For example, both flavonoids and isoflavonoids
   could protect plants against herbivorous insects by negatively
   affecting their behavior, growth, and development [[43]10,[44]11].
   Flavonoids affected the nutrient accumulation in Piezodorus guildinii
   nymphs, making them unable to complete the molting process and causing
   death [[45]12]. The addition of coumarin and cinnamic acid to the diet
   of Helicoverpa armigera larvae and Spodoptera litura larvae
   significantly increased the mortality of the tested insects [[46]13].
   In addition, flavonoids can inhibit digestion in insects as feeding
   deterrents [[47]14]. Four flavonoids, isoglabratephrin,
   (+)-glabratephrin, tephroapollin-F, and lanceolatin-A, reduced the
   relative growth rate, relative consumption rate, and efficiency of
   conversion of ingested food by three stored grain insects [[48]15].

   Allium plants possess a variety of nutritional, biological, and
   health-promoting properties, including antimicrobial, antioxidant,
   anti-tumor, immunoregulatory, antidiabetic, and anti-inflammatory
   effects [[49]16]. Some studies have indicated that a total of 23
   flavonoids are identified from Allium mongolicum, among which the
   content of rutin and isoquercitrin was relatively large
   [[50]17,[51]18,[52]19]. The sugar residues in Allium spp. are mainly
   composed of linear or branched glucose, rhamnose, galactose, xylose,
   and arabinose units [[53]20]. Galactose is a significant nutrient for
   insect herbivores [[54]21]. Rutin is a flavonoid found predominantly in
   different plant parts and exists as a glycoside
   (3-O-rhamnoglucoside/rutinose) of quercetin [[55]22]. Rutin is shown to
   possess antioxidant activity [[56]23]. Rutin and quercetin-3-glucoside
   suppressed the development and increased the mortality of Lymantria
   dispar [[57]14]. Rutin prolonged the developmental duration of
   Spodoptera frugiperda [[58]24] and S. litura larvae [[59]25]. Quercetin
   increased the development time, pre-reproductive period, and mortality
   and reduced fecundity in Acyrthosiphon pisum [[60]26], and it also
   negatively affected larval growth and pupal weight in S. litura
   [[61]27]. However, quercetin was not yet reported as a phagostimulant
   [[62]28].

   Herbivorous insects can tolerate these toxic secondary substances
   because they have evolved various physiological mechanisms to avoid
   their harmful effects. Most insect herbivores depend heavily on
   enzymatic degradation for the neutralization of ingested plant
   secondary metabolites. Main detoxifying enzymes include cytochrome P450
   (CYP450), glutathione S-transferase (GST), and carboxylesterase (CarE),
   among which CYP450 enzymes are the most extensively studied enzymes
   that effectively metabolize a wide variety of toxic substances
   [[63]29,[64]30]. Insect CYP450 genes can be induced by a variety of
   plant secondary metabolites [[65]31]. In Depressaria pastinacella,
   CYP450 activity rose in response to the presence of furanocoumarins in
   its diet [[66]32]. After injecting a tea saponin solution into the
   abdomen of adult Locusta migratoria, the activity of CYP450, GST, and
   CarE significantly increased [[67]33]. Nevertheless, the molecular
   mechanism of herbivorous insects’ detoxification or tolerance to
   secondary plant compounds remains unclear.

   Galeruca daurica (Joannis) belongs to Coleoptera, Chrysomelidae
   ([68]Figure 1). It is reported to be mainly distributed in Mongolia,
   Russia (Siberia), North Korea, South Korea, and China, including Inner
   Mongolia, Xinjiang, and Gansu [[69]34]. G. daurica has been a new pest
   in the grasslands of Inner Mongolia since its abrupt outbreak in 2009
   [[70]35]. This pest is an oligophagous herbivore, only feeding on
   Allium plants of the Liliaceae family, among which A. mongolium is the
   most suitable host plant [[71]35]. Therefore, G. daurica must have the
   ability to detoxify or tolerate the main secondary metabolites in
   Allium plants. Our previous study showed that six metabolites in A.
   mongolium, including three flavonoids (isoquercitrin, isoflavone, and
   rutin) and three monosaccharides (β-d-glucopyranose, d-galactose, and
   l-rhamnose), affected the feeding preference of G. daurica [[72]36].
   For example, the addition of 0.1 mM isoflavones decreased the feed
   intake of larvae, and the feeding preference index decreased
   significantly. However, its mechanism of detoxification or tolerance
   remains unknown. In this study, these six metabolites in A. mongolium
   were respectively added to the diet for the third instar larvae of G.
   daurica. The purpose was to screen out the differentially expressed
   genes (DEGs) by performing high-throughput transcriptome sequencing and
   analyzing the detoxification metabolic pathways of this pest to host
   secondary metabolites so as to lay a foundation for further exploring
   the molecular mechanism of G. daurica adaptation to host plants.

Figure 1.

   [73]Figure 1
   [74]Open in a new tab

   Pictures of G. daurica morphology and damage. (a) larvae; (b) adults;
   (c) damaged grassland.

2. Materials and Methods

2.1. Insects

   The 1st and 2nd instar larvae were collected from the Siziwang
   grasslands of Inner Mongolia (42.2012° N, 112.0894° E), China, on 7 May
   2023, and reared at 26 ± 1 °C, 60~80% relative humidity under a 16 h
   light/8 h dark period in a constant temperature incubator. The 3rd
   instar larvae were reared on Allium tuberosum for experiments.

2.2. Sample Processing

   A total of 10% dimethyl sulfoxide (DMSO) solution was prepared with
   distilled water, which was used to prepare 0.1 mmol/L isoquercitrin
   (IQ), isoflavone (ISO), rutin (RT), d-galactose (Gal),
   β-d-glucopyranose (Glc), and l(+)-rhamnose (Rham) treatment solutions.
   Information on chemical substances and solvents is shown in [75]Table
   S1.

   A total of 7 experimental groups were set up, including 6 treatment
   groups and 1 solvent control group. Each insect box contained 20 3rd
   instar larvae of G. daurica. For the treatment groups, larvae fed on A.
   tuberosum soaked in 6 kinds of treatment solution for 5 min,
   respectively. For the solvent control group, larvae fed on the leaves
   of A. tuberosum soaked in 10% DMSO solution every day. Three days after
   feeding, 10 larvae in each insect box were collected for transcriptome
   sequencing. Three biological replicates were set up in each
   experimental group.

2.3. cDNA Library Construction and Transcriptome Sequencing

   Transcriptome sequencing was performed by Ovison Gene Technology Co.,
   Ltd. (Beijing, China). A total of 21 cDNA libraries were constructed,
   including 7 groups with 3 times per 10 larvae. RNA purity (OD260/280
   and OD260/230) and RNA fragment length were measured by Nanodrop
   (Thermo Scientific, Thermo Fisher Scientific, Waltham, MA, USA) and
   Agilent 2100 (Agilent Technologies, Santa Clara, CA, USA) prior to
   library construction, and transcriptome sequencing was performed after
   qualification. The constructed libraries were sequenced using the
   Illumina Novaseq 6000 high-throughput sequencing platform, the raw data
   obtained by sequencing were filtered to remove the joint sequences and
   low-quality reads, and the high-quality sequences were finally
   obtained. Trinity v2.4.0 software [[76]37] was used to splice and
   assemble clean read sequences to obtain unigenes.

2.4. Analysis and Annotation of Differentially Expressed Genes

   DEseq1.10.1 software [[77]38] was used to analyze the differentially
   expressed genes (DEGs), and the p value was corrected by the
   Benjamini–Hochbery (BH) method. The final corrected p value, i.e., the
   false discovery rate (FDR), was used as the key index for the screening
   of DEGs. Fold change (FC) represented the ratio of gene expression
   between samples of each treatment (IQ, ISO, RT, Gal, Glc, and RT) and
   control (DMSO). FDR < 0.05 and |log[2](FC)| ≥ 1 were used as the
   screening criterion for DEGs. BLAST was used to compare the DEG
   sequences with Nr, Swiss-Port, GO, KOG, Pfam, and KEGG databases (E
   value < 1 × 10^−5) to obtain the corresponding annotation information.
   GOseq v1.22.0 was used for enrichment analysis [[78]39], the Cluster
   Profiler was used for KEGG pathway enrichment analysis, and the
   corrected p value (q) < 0.05 was used as the significantly enriched
   KEGG pathway.

2.5. Verification of Differentially Expressed Genes by qRT-PCR

   Seven DEGs screened in [79]Section 2.4 were randomly selected for
   qRT-PCR verification, including glutathione S transferase (GST,
   TRINITY_DN40461_c0_g1), pupal cuticle protein Edg-84A-like protein (CP,
   TRINITY_DN48195_c0_g1), elongation factor 1 alpha (ef1α,
   TRINITY_DN50797_c3_g2), cytochrome P450 insecticide
   resistance-associated cytochrome P450 (CYP450, TRINITY_DN47138_c1_g1),
   glycoside hydrolase family protein 48 (GH48, TRINITY_DN28963_c0_g1),
   chemosensory protein 2 (CSP2, TRINITY_DN44890_c0_g1), and
   peptidoglycan-recognition protein-SC2 (PGRPSC2, TRINITY_DN48140_c0_g2).

   qRT-PCR was performed to confirm the reliability of the RNA sequencing
   results. The same samples were used as templates for sequencing. The
   primers were designed using the Primer Premier3.0
   “[80]https://bioinfo.ut.ee/primer3-0.4.0/ (accessed on 19 March 2024)”
   and synthesized by Sangon Biotech (Shanghai, China) ([81]Table S2).
   qRT-PCR was conducted on the FTC-3000 (Funglyn Biotech, Toronto,
   Canada) using the MonAMP^TM SYBR^® Green qPCR Mix (Monad, Wuhan,
   China). The reaction procedure was as follows: 95 °C for 10 min
   followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. The
   reaction procedure of the melting curve was 95 °C for 15 s, 60 °C for
   15 s, and 95 °C for 15 s. Three technical replicates of each reaction
   were conducted, and SDHA was used as a reference gene [[82]40]. The
   2^−ΔΔCt method was used to calculate the relative expression levels
   [[83]41].

3. Results

3.1. The Basic Characteristics of Transcriptome Sequencing Analysis

   In order to clarify the effects of host plant metabolites on the gene
   expression in G. daurica larvae at the transcriptome level, twenty-one
   sequencing libraries were constructed from seven groups of the third
   instar larvae fed on the diet containing different metabolites. The raw
   data have been deposited in the National Center for Biotechnology
   Information (NCBI) under BioProject ID PRJNA1093491. A total of
   472,847,958 raw read pairs were measured, and 469,646,761 clean read
   pairs were obtained after quality control. The clean data of each
   sample were 5.25~7.05 Gb, the G + C content was 34.56~37.13%, the error
   rate was 0.02~0.03%, the Q20 base ratio was 97.89~98.28%, and the Q30
   base ratio was 93.78~94.76% ([84]Table S3). A total of 124,348 unigenes
   were obtained, and N50 was 1127 bp. Among them, the number of sequences
   with a splicing length of 300 to 500 bp was 61,079, the number of
   sequences from 500 to 1000 bp was 36,605, the number of sequences from
   1000 to 2000 bp was 16,919, the number of sequences greater than 2000
   bp was 9745, and there were no sequences less than 300 bp ([85]Figure
   S1). These results indicate that the transcriptome data are reliable
   and can be used for subsequent analysis.

3.2. Analysis and Annotation of Differentially Expressed Genes (DEGs)

   In order to screen out the DEGs between different treatments and the
   control, the expression data were statistically analyzed. The results
   showed that compared with the control, a total of 291 DEGs were
   detected after larvae were fed on the diet containing six different
   metabolites, respectively, and the treatment groups of isoquercitrin,
   isoflavone, rutin, d-galactose, β-d-glucopyranose, and l-rhamnose had
   110, 29, 6, 17, 40, and 43 up-regulated DEGs and 20, 5, 23, 4, 32, and
   54 down-regulated DEGs, respectively ([86]Figure 2). In order to better
   reflect the effects of feeding different substances on the
   transcriptional level in larvae, a Venn diagram was drawn with FPKM > 1
   as the criterion for judging gene expression ([87]Figure 3). In primary
   metabolite treatment groups, the l-rhamnose treatment group contained
   the highest number of DEGs (97), and the d-galactose treatment group
   contained the lowest (21). In secondary metabolite treatment groups,
   the isoquercitrin treatment group contained the highest number of DEGs
   (130), and the rutin treatment group contained the lowest (29).

Figure 2.

   [88]Figure 2
   [89]Open in a new tab

   Statistical map of the gene number of the differentially expressed gene
   set. DMSO (control group): 10% dimethyl sulfoxide; IQ, ISO, RT, Gal,
   Glc, and RT (treatment groups): 0.1 mM isoquercitrin, isoflavone,
   rutin, d-galactose, β-d-glucopyranose, and l(+)-rhamnose, respectively.
   Three replicates were set up in each experimental group, and each
   repeat included 10 larvae.

Figure 3.

   [90]Figure 3
   [91]Open in a new tab

   Venn diagram of differentially expressed genes. Red outline area: IQ
   vs. DMSO; orange outline area: ISO vs. DMSO; yellow outline area: RT
   vs. DMSO; green outline area: Glc vs. DMSO; cyan outline area: Gal vs.
   DMSO; blue outline area: Rham vs. DMSO.

   The DEGs are listed in [92]Table S4. Among them, there were twenty-six
   major DEGs (FDR < 0.01 with functional annotations) in the
   isoquercitrin treatment group (twenty-two up-regulated and four
   down-regulated). The up-regulated genes mainly included dehydrogenase,
   glycoside hydrolase family protein 48, cytochrome P450, cytochrome b,
   glycoside hydrolase family 1, transposase, and solute carrier family 13
   member 3. The down-regulated genes included elongation factor 1-alpha,
   C-type lectin precursor, coat protein, and cytochrome c oxidase subunit
   I. There were fifteen major DEGs in the isoflavone treatment group
   (fourteen up-regulated and one down-regulated). The up-regulated genes
   mainly included ribosomal proteins, solute carrier family 13 member 3,
   glyceraldehyde-3-phosphate dehydrogenase, cytochrome b, and cytochrome
   c oxidase subunit II. The down-regulated gene was cytochrome c oxidase
   subunit I. There were seven major DEGs in the rutin treatment group
   (three up-regulated and four down-regulated). The up-regulated gene was
   Ferritin, and the down-regulated genes included cytochrome c oxidase
   subunit I, peptidoglycan-recognition protein-SC2, polyprotein, and 31
   kDa antigen. In the β-d-glucopyranose treatment group, there were 24
   major DEGs (10 up-regulated and 14 down-regulated). The up-regulated
   genes mainly included Chlorophyll a-b-binding protein 40, reverse
   transcriptase homolog, and solute carrier family 13 member 3. The
   down-regulated genes mainly included 31 kDa antigens, hemoglobin
   subunit beta, peptidoglycan-recognition protein-SC2, cytochrome c
   oxidase subunit I, elongation factor 1-alpha 1-like protein,
   Kruppel-like protein 1, and Ferritins. In the l-rhamnose treatment
   group, there were 40 major DEGs (25 up-regulated and 15
   down-regulated). The up-regulated genes mainly included cytochrome c
   oxidases subunit I, heat shock proteins, glyceraldehyde-3-phosphate
   dehydrogenase, and ribosomal proteins. The down-regulated genes mainly
   included 31 kDa antigen, hemoglobin subunit beta, attacin-like immune
   proteins, peptidoglycan-recognition protein-SC2, and defensin 1
   precursor. In the d-galactose treatment group, there was only one
   up-regulated major gene (phosphoserine aminotransferase).

3.3. GO and KEGG Enrichment Analysis of Differentially Expressed Genes (DEGs)

   To further understand the function of DEGs, these DEGs were subjected
   to GO and KEGG enrichment analysis. GO enrichment showed that most GO
   terms were enriched in biological processes and molecular functions,
   and there were fewer in cellular components ([93]Figure 4). In the
   isoquercitrin treatment, most DEGs were significantly enriched in
   catalytic activity, and, secondly, oxidoreductase activity and
   carbohydrate metabolic process. In the isoflavone treatment, most DEGs
   were significantly enriched in cellular components. For molecular
   functions, most DEGs were significantly enriched in oxidoreductase
   activity, and, secondly, structural constituent of ribosome. For
   biological processes, most DEGs were significantly enriched in the
   generation of precursor metabolites and energy, and, secondly, ATP
   metabolic process and cation transport. In the rutin treatment, most
   DEGs were significantly enriched in GO terms related to defense and
   immune responses. In the d-galactose treatment, the structural
   constituent of the cuticle was only one significantly enriched GO term.
   In the β-d-glucopyranose treatment, most DEGs were significantly
   enriched in the cytoplasm, and, secondly, the cytoplasmic part. For
   molecular functions, oxidoreductase activity was the GO term
   significantly enriched by most DEGs, and secondly, iron ion binding.
   For biological processes, nearly all DEGs were significantly enriched
   in GO terms related to ion transport and homeostasis. In the l-rhamnose
   treatment, most DEGs were also significantly enriched in the cytoplasm,
   and, secondly, the cytoplasmic part. For biological processes, nearly
   all DEGs were significantly enriched in GO terms related to various
   responses and transmembrane transports. For molecular functions, most
   DEGs were significantly enriched in electron transfer activity.

Figure 4.

   [94]Figure 4
   [95]Open in a new tab

   GO enrichment analysis of differentially expressed genes in the larva
   of G. daurica. (a–f) indicate isoquercitrin, isoflavone, rutin,
   d-galactose, β-d-glucopyranose, and l(+)-rhamnose 6 different
   treatments, respectively. *: significant enrichment with p value (q) <
   0.05.

   KEGG enrichment analysis showed that most DEGs were enriched in various
   metabolism pathways ([96]Figure 5). For all six metabolites, glycine,
   serine, and threonine metabolism was only one commonly enriched KEGG
   pathway. Common enriched KEGG pathways were carbon metabolism and
   pyruvate metabolism for three primary metabolites and three secondary
   metabolites, respectively. In the isoquercitrin treatment, the pathways
   with the highest enrichment intensity included vitamin B6 metabolism,
   nicotinate and nicotinamide metabolism, glycine, serine, and threonine
   metabolism, and one carbon pool by folate. In the isoflavone treatment,
   the pathways with the highest enrichment intensity included
   glycolysis/gluconeogenesis, pyruvate metabolism, ribosome, and
   biosynthesis of amino acids. In the rutin and d-galactose treatments,
   fewer DEGs were enriched in fewer KEGG pathways, and the pathways with
   the highest enrichment intensity were the pentose phosphate pathway in
   the rutin treatment and vitamin V6 metabolism in the d-galactose
   treatment. In the β-d-glucopyranose treatment, the pathways with the
   highest enrichment intensity included glycolysis/gluconeogenesis and
   the pentose phosphate pathway. In the l-rhamnose treatment, the
   pathways with the highest enrichment intensity included taurine and
   hypotaurine metabolism, ECM–receptor interaction,
   glycolysis/gluconeogenesis, and pyruvate metabolism.

Figure 5.

   [97]Figure 5
   [98]Open in a new tab

   KEGG enrichment analysis of differentially expressed genes in the larva
   of G. daurica. (a–f) indicate isoquercitrin, isoflavone, rutin,
   d-galactose, β-d-glucopyranose, and l(+)-rhamnose 6 different
   treatments, respectively.

3.4. Validation of Differentially Expressed Genes (DEGs)

   According to the results of transcriptome DEG analysis, seven DEGs
   (GST, CP, ef1α, CYP450, GH48, CSP2, and PGRPSC2) were randomly selected
   for qRT-PCR verification ([99]Figure 6). The results showed that
   qRT-PCR and RNA-seq trends of seven DEGs were completely consistent,
   indicating that the transcriptome sequencing results of this experiment
   are reliable.

Figure 6.

   [100]Figure 6
   [101]Open in a new tab

   Validation of RNA-seq-derived changes in transcript abundance with
   qRT-PCR for 7 differentially expressed genes. Fold change:
   normalization values RNA sequencing data and relative expression values
   obtained with qRT-PCR based on the 2^−ΔΔCt method. (A) Isoquercitrin;
   (B) isoflavone; (C) rutin; (D) d-galactose; (E) β-d-glucose; (F)
   l-rhamnose.

4. Discussion

   Herbivorous insects have a very close relationship with host plants,
   which constitute the food source, mating site, oviposition site, and
   habitat of all or part of their life cycle, and the digestion and
   utilization of nutrients (plant primary metabolites) and the adaptation
   of secondary metabolites are the key factors for insects to establish
   populations [[102]42]. In this study, after the larvae of G. daurica
   fed, respectively, on foods containing six different plant metabolites
   (three primary metabolites and three secondary metabolites), most DEGs
   were enriched in various metabolic pathways, suggesting that host
   plants may affect the biology of herbivorous insects by mainly
   mediating their various metabolic processes. Glycine, serine, and
   threonine metabolism was the only common enriched KEGG pathway by the
   DEGs among the six metabolite treatments, indicating that both primary
   and secondary metabolites can affect amino acid metabolism. Carbon
   metabolism was the only common enriched KEGG pathway by the DEGs among
   the three primary metabolites (carbohydrates), showing that
   carbohydrates in host plants mainly affect the absorption,
   transportation, decomposition, and synthesis of carbon in herbivorous
   insects. Plant secondary metabolites can affect insect feeding, growth
   and development, and reproduction, and even kill insects with toxicity
   [[103]43]. The adaptation process of insects to plant secondary
   metabolites requires the participation of a variety of enzymes, and
   feeding on foods containing different secondary metabolites will affect
   the expression of genes. Six CYP450 genes and two esterase genes were
   up-regulated in the isoquercitrin treatment for this paper, which is
   similar to the results of others. For example, in Spodoptera
   frugiperda, CYP450 activity was induced to increase by a variety of
   secondary metabolites, such as flavonoids, menthol, or
   indole-3-carbinol, and was also involved in the metabolism of multiple
   secondary metabolites such as indole, coumarin, flavonoids, and
   glucosinoside [[104]30]. Ferulic acid induced the expression of GST
   family genes in Sitodiplosis mosellena [[105]44].

   Cytochrome b is a component of the mitochondrial respiratory chain, is
   involved in electron transport in the respiratory chain, and is closely
   related to cellular energy metabolism [[106]45]. Cytochrome b is
   oxidized by the cytochrome c oxidase, which finally converts molecular
   oxygen (O[2]) to water (H[2]O) [[107]46]. In the present study,
   multiple genes encoding cytochrome b and cytochrome c oxidase (COI and
   COII) were differentially expressed in the other treatment groups,
   except for d-galactose, especially two COI, two COII, and two
   cytochrome b in the l(+)-rhamnose treatment and two COI, one COII, and
   one cytochrome b in the isoflavone treatment, which suggests that these
   plant metabolites may have an important effect on energy metabolism in
   G. daurica.

   l-rhamnose is a monosaccharide that is widely present in nature,
   especially in some plants or microbial polysaccharides with anti-tumor
   activity, and it has functions such as regulating carbohydrate and
   lipid metabolism in animals [[108]47,[109]48,[110]49]. In the
   l-rhamnose treatment, we found that the down-regulated genes mainly
   included 31 kDa antigen, attacin-like immune proteins,
   peptidoglycan-recognition protein-SC2, and defensin 1 precursor, which
   suggests that l-rhamnose may be related to the immune defense in G.
   daurica.

   Ribosome is a kind of ribonucleoprotein particle in biological cells
   whose main function is to synthesize amino acids into protein
   polypeptide chains with mRNAs as the guide, and it is an important
   organelle for protein synthesis in cells [[111]50]. In this study, a
   large number of ribosomal protein genes were up-regulated in the
   isoflavone and l-rhamnose treatments, which implies that isoflavone and
   l-rhamnose may have significant effects on protein synthesis in G.
   daurica.

   In summary, the addition of six different substances to the diet mainly
   affected the ribosome and metabolic pathways of G. daurica, and the
   adaptation of larvae to allium host may be through the synthesis of
   corresponding proteins and the acceleration of metabolism.

5. Conclusions

   A total of 291 DEGs were identified compared to the solvent control
   (DMSO). The main DEGs in the isoquercitrin, isoflavone, rutin,
   d-galactose, β-d-glucopyranose, and l-rhamnose treatment groups were
   130, 34, 29, 21, 72, and 97, respectively. GO and KEGG enrichment
   analysis showed that most DEGs were enriched in various metabolic
   pathways, implying that these six main primary and secondary
   metabolites in Allium plants may affect various metabolic processes in
   the larvae of G. daurica.

Acknowledgments