Identification and functional analysis of diet-responsive genes in Spodoptera litura (Fabricius)

Background: Spodoptera litura is one of the most devastating agricultural pests with a wide range of host plants. To study larval performance on different diets and midgut adaptation at transcriptional levels, feeding assay and RNA-Seq experiments were conducted. RNA interference technology was used to explore the detoxification and metabolism of two cytochrome P450 genes. Results: The bioassay data showed that Spodoptera litura larvae developed more quickly when fed on cabbage than when fed on soybean, corn and cotton, tannin can inhibit the growth of Spodoptera litura . The result of RNA-Seq indicated that Spodoptera litura midgut modified gene expression levels to accommodate different diets, and the most differentially expressed genes were detoxification-related and digestion-related genes. Further analysis showed that the glutathione metabolism pathway was the common detoxification pathway in Spodoptera litura. The expression of cytochrome P450 genes showed a clear response to different plant hosts, and these differences may play key functions in primary detoxification of secondary metabolites from host plants. Meanwhile, the digestive enzymes of proteinases, lipases, and carbohydrases in midgut showed special responses to different plant hosts. After injection of dsRNA of CYP321A19 and CYP6AB60 , the expression level of target gene were decreased, and the sensitivity of insect to plant allelochemicals increased and the weight increase significantly slowed. Conclusion: In this study, genes involved in detoxification were identified, and the results demonstrate the genes and pathways Spodoptera litura utlize to detoxify specific plant-host allelochemicals. These results may also provide a theoretical basis for Spodoptera litura management. current study was to determine global changes in the midgut gene expression of S. litura larvae fed on different diets, as well as to compare the performance of S. litura larvae when fed on these diets. To investigate these transcriptional responses, we used a replicated RNA-Seq approach. In our analyses of the transcriptional responses of S. litura larval midgut, we focused mainly on the digestion-related and detoxification-related genes, with a particular emphasis on cytochrome P450 genes. In addition, two P450 genes were selected in the transcriptome, and the function was verified by RNA interference technology.


Growth Rate On Different Diets
The larvae grew differently on the different diets. The initial weight of S. litura transferred to cabbage was significantly higher than those fed on the soybean and Tannin1 (T 0 ). Two days later (T 1 ), the weight of S. litura fed on cabbage was 61.02 mg, which was significantly heavier than those fed on other diets, and especially for Tannin1 (29.91 mg).
At the last time point (T 3 ), the average weight of S. litura fed on cabbage (520.37 mg) was heaviest.
Those larvae fed on soybean (365.38 mg), cotton (272.09 mg), and artificial diet (193.25 mg) had a middle range weight, and those fed on corn (91.01 mg), Tannin2 (49.24 mg), and Tannin1 (36.98 mg) were significantly lighter than the others (Fig. 1). Larvae fed on cabbage showed the highest growth rate at time points 1 and 2, and the lowest growth rate was found for the larvae fed on the artificial diet with15 g of tannin at any time point (Table 1). S. litura larvae had a higher growth rate and a higher final weight when fed on cabbage. S. litura larvae fed on Tannin1 had growth rates and final weights that were significantly lower. Table 1 Growth rate of S. litura larvae fed on different diets.
Illumina Sequencing, Sequence Assembly, And Gene Identification Transcriptome analysis of 21 samples was completed, and clean reads were obtained. The percentage of Q30 bases was not less than 91.22%. Clean reads of each sample were sequenced with the designated reference genome of S. litura [28], and the alignment efficiency ranged from 83.84-89.66%. A total of 570 new genes were identified by filtering out sequences that were either too short (less than less than 150 nucleotide ORFs) or contained only a single exon. Sequence alignment of the new genes was conducted using BLAST software with the databases of NR, swiss-prot, GO, COG, KOG, Pfam, and KEGG. KEGG Orthology results of the new genes were obtained using KOBAS2.0 [29]. After the prediction of amino acid sequences of the new genes, the HMMER [30] software was used to compare our results with the database of Pfam to obtain the annotation information of the new genes ( Table 2). The transcriptome sequences had submitted to NCBI database and the SRA accession number was PRJNA528696  Fig. 2).
The three KEGG pathways of metabolism of xenobiotics by cytochrome P450, drug metabolism cytochrome P450, and glutathione metabolism were mostly up-regulated in cabbage, corn, cotton, and soybean feed samples as compared to artificial diets, but were down-regulated in the artificial diet with tannin feed samples compared to the artificial diet alone.

The Character Of Genes Involved In Enriched Pathway
In up-regulated pathway, there were many similar genes involved in different pathways. Glutathione S-transferase and UDP-glucuronosyltransferase were two important genes present in up-regulated pathway. Both of these genes are important secondary metabolism detoxification enzymes in insects.
Glutathione S-transferase was present in three pathways out of the top 5 pathways determined in our analysis. We observed metabolism of xenobiotics by cytochrome P450, Drug metabolism-cytochrome P450 and Glutathione metabolism, and because metabolism of xenobiotics by cytochrome P450 and drug metabolism -cytochrome P450 were common in vertebrate animals and few reports in insect, and no cytochrome P450 genes were involved in those pathways, it indicated that glutathione metabolism was the main pathway, in which Glutathione S-transferase was involved.
A total of 17 glutathione S-transferase genes, 1 gamma-glutamyltransferase (GGT), 3 glutathione peroxidases (GPx), and 1 isocitrate dehydrogenase (IDH) gene were involved in the glutathione metabolism pathway. Generally, no special glutathione S-transferase genes were be found when insects were fed with different host-plants, which indicated that Glutathione metabolism pathway were the common detoxification pathway in S. litura. Meanwhile, elevated expression of genes involved in glutathione disulfide produced in feed on cabbage and cotton samples was observed, and gamma-glutamyltransferase genes were elevated when insects were fed on cotton (Supplement Fig. 3).

Identification Of Cytochrome P450s Related To Detoxification
As our focus was primarily on the response of detoxification-related genes of S. litura fed on various diets, we paid special attention to the cytochrome P450 gene family, which is involved in primary detoxification metabolism. The main cytochrome P450 gene involved in detoxification metabolism in insects is typically the special cytochrome P450. However, in the enriched up-regulated pathways from our analysis, no cytochrome P450 genes were present.
A total of 24 cytochrome P450 genes of which FPKM > 100 were chosen in S. litura midgut fed on plant hosts. Of these, 19 out of 24 cytochrome P450 genes belonged to the CYP6 family. Considering the cytochrome P450 genes found in the CYP family: 2 belonged to CYP4, 2 belonged to CYP9, and 1 belonged to CYP12. Unlike in Glutathione metabolism pathway, the expression of cytochrome P450 genes showed a clear response to different plant hosts. There were more induced cytochrome P450 genes when S. litura fed on cabbage and cotton than on other diets (12 genes associated with cabbage and 9 genes associated with cotton). Only 3 cytochrome P450 genes were higher expressed when fed on artificial diet, 2 genes when fed on soybean, and 1 gene when fed on corn.
Artificial diets containing tannin induced 2 cytochrome P450 genes to be expressed in insects, but suppressed the expression of 2 cytochrome P450 genes which had higher expression when feed on artificial diets alone (Supplement Fig. 4).
Expression pattern of CYP321A19 and CYP6AB60 in different developmental stages and tissues In order to obtain the expression profile of CYP321A19 and CYP6AB60, RT-qPCR analysis showed that CYP321A19 and CYP6AB60 transcript was detected in all tissues and age. For CYP6AB60 gene, it is highly expressed at the 4th and 6th instar larva, and the expression level is lower at the 1st instar larva and pupa ( Fig. 2A), and the expression levels were significantly higher in the midgut and fat body ( Fig. 2B). Similarly, CYP321A19 was also highly expressed in 4th instar larvae ( Fig. 2C), with the highest expression in fat body and midgut (Fig. 2D).

Expression of CYP321A19 and CYP6AB60 was induced by plant allelochemicals
The expression of CYP321A19 in the midgut and fat body of the larvae was increased and showed a significant difference with control artificial diet, when fed with an artificial diet containing quercetin When the larvae were exposed to the plant allelochemicals, the net weight gain on day 5 was lower in the treatment group than in the control group (CYP321A19: 0.57 g vs. 0.70 g) (Fig. 5A). Similarly, daily weight gain was lower in the treatment group than in the control group (Fig. 5B).Thus, CYP321A19 silenced larvae showed both net weight gain and daily growth significantly lower than the control group. In addition, larvae injected with dsCYP321A19 and fed with coumarin and soy isoflavones, exhibited significantly lower weight gains than dsGFP-injected controls exposed to the same allelochemicals ( Fig. 5C-F).

Identification Of Digestive Enzymes Related To Diet Adaption
When S. litura fed on different diets, it faced different secondary metabolism stresses when deal with different nutrients. Proteinases, lipases, and carbohydrases make up the main digestive enzymes of insects [31]. In our transcriptome data, digestive enzymes in midgut were identified, and included proteinases (trypsin and chymotrypsin), lipases, and carbohydrases (alpha-amylase and glucosidase).
A total of 34 trypsin genes and 4 chymotrypsin genes were found to be more highly expressed in S.
litura midgut (FPKM > 100). We found more than 10 induced trypsin genes when S. litura fed on cotton, soybean, and artificial diet, but few trypsin genes were induced when fed on cabbage and corn. Most of the high expression trypsin genes were uniquely induced by diets. We found 8, 11, and 6 unique high expression genes when fed on artificial diet, soybean, and cotton, respectively, and 5 higher expression trypsin genes were induced when fed on cotton and soybean. Most high expression chymotrypsin genes were only detected in samples fed on artificial diets, cotton, and soybean.
Considering the lipid digestion and absorption process in the midgut, 13 higher exressing triacylglycerol lipase genes were (FPKM > 100) were found. The triacylglycerol lipase genes were induced when insects were feed on corn, cotton, and soybean, and had the highest induced gene numbers when fed on soybean (triacylglycerol lipase genes). When fed on cotton, a total of 6 triacylglycerol lipase genes were induced. All the corn-induced triacylglycerol lipase genes were the same as those induced by soybean, except LOC111355064. There were 2 cotton-induced triacylglycerol lipase genes that were the same as soybean-induced genes, but there were no shared triacylglycerol lipase genes with corn.
During carbohydrate digestion and absorption process in the S. litura midgut, amylases and glucosidases were the main observed differentially expressed genes. A total of 2 amylase and 12 glucosidase genes showed higher expression (FPKM > 100). We found that 1 alpha-amylase was induced in corn and cotton fed insects. However, there were no observed induced alpha-amylase genes in other diet treatments. In corn and cotton fed samples, there was 1 alpha-amylase gene with higher induced expression. No other diets showed induced alpha-amylase gene expression (Supplement Fig. 5).

Discussion
Insect herbivores can feed on their host plants for development and survival. Due to host plant nutrition and allelochemicals, polyphagy insects show differential fitness to the plant-hosts [32].
Studies have shown that plant secondary metabolites can have positive effects on the survival and growth rates of insects [33,34,35]. Our larval development assays suggested that cabbage is the best host plant for S. litura, with a higher growth rate and final weight than other host plants. S. litura fed on artificial diets with tannin had a lower growth rate and obtained a lighter final weight in our study.
In the study, we chose host-plants that contain various kinds of secondary metabolites. Gossypol, tannin, and flavonoids are the major secondary metabolites in cotton plants, and glucosinolates and isoflavones are rich in Chinese cabbage and soybean. Derivatives of 1, 4-benzoxazin-3-one are the common secondary metabolites found in maize plants. The explanation for the differences of larval development on different diets could be due to the effect of differential secondary metabolites that the diets contain. A major question in plant-insect interactions is how insect herbivores cope with secondary metabolites compounds in diverse host plants [36]. As insect midgut is the main location to digestive food and detoxification [37], and the mechanisms S. litura use to cope with toxic compounds in diverse host plants are not well understood [38], we used a feeding assay and RNA-Seq of S. litura larval midgut to determine the genes used by S. litura to cope with secondary metabolites associated with different diets.
Based on S. litura genomic data [28], high-throughput sequencing was an efficient research tool to better understand the molecular mechanisms behind adoption of host-plants. Our results demonstrated that S. litura could develop on different diets and the transcriptional responses of midgut were related to the host diet that S. litura was fed on. Generally, glutathione S-transferase genes and Cytochrome P450 genes were the most differential expressed genes involved in detoxification, while, proteinases, lipases, and carbohydrases were the most differentially expressed genes involved in the digestive system.
A total of 47 Glutathione S-transferase genes were identified in S. litura genomic data [28]. In this study, 17 glutathione S-transferase genes were found to be high expressing in S. litura midgut when fed on different host-plant. It has been demonstrated that glutathione S-transferase can detoxify many plant allelochemicals, and can be induced by plant allelochemicals [21,39]. In this study, the expression of some glutathione S-transferase genes showed high FPKM valves, but no special response glutathione S-transferase genes were be found when fed with different diets, which indicated that glutathione metabolism pathways were the common detoxification pathway in S. litura.
At same time, genes in glutathione disulfide produced in feed on cabbage and cotton samples also showed high FPKM valves. One possible reason for this is that more glutathione disulfide was needed for allelochemical detoxification. As the gamma-glutamyltransferase genes, which are located at last step of glutathione metabolism pathway, were higher expressed in insects fed on cotton, cotton allelochemical detoxification may require more steps than detoxification in S. litura midgut fed on other host-plants.
In insects, cytochrome P450 genes are an important gene family in the detoxification of exogenous or endogenous compounds, including plant secondary metabolites [40,41]. A total of 138 cytochrome P450 genes were identified in S. litura genomic data [28]. In this study, 17 glutathione S-transferase genes were highly expressed in S. litura midgut when fed on different host-plants. 21 out 61 Clan 3 cytochrome P450 genes were highly expressed in S. litura midgut when fed on different host-plants, and most of these genes belong to the CYP6 gene family. In insect, the CYP6 gene family is mostly involved in plant secondary metabolites detoxification. Here, we selected two P450 genes from the transcriptome and named them CYP6AB60 and CYP321A19. They all showed the highest expression levels in the 4th instar larva, and the highest expression levels in the 4th instar larva midgut and fat body. When the larvae were exposed to an artificial diet containing quercetin or coumarin and soy isoflavones, the expression levels of the selected genes were significantly up-regulated. Similar results have been reported that CYP6AE14 and CYP6AE11 were significantly up-regulated when H.
armigera fed on artificial diet with higher concentration of gossypol [42]. CYP6B46 can be induced when Manduca sexta larvae feed on Nicotiana wild-type plants, which can produce nicotine [43]. The expression of CYP314A1, CYP315A1, CYP18A1, CYP307A1, and CYP306A1 were found to be induced by 2-tridecanone [44]. The xanthotoxin can induce the expression of CYP9A genes from larval Manduca sexta midgut [45]. The above research results are similar to the results of this study.
In the detoxification of plant secondary metabolites, cytochrome P450 can be classed into specialists and generalists [21]. In this study, most high expressing cytochrome P450 genes may be specialists to different host-plant, and those cytochrome P450 gene family may be involved in primary detoxification metabolism. As specialists have a highly efficient and specialized detoxification system, S. litura has a wide host range and significantly impacts agricultural production. The specialist cytochrome P450 genes may represent a potential target site for the development of pest controls.
RNAi technology has been widely used to reveal the role of cytochrome P450 in drug resistance, secondary metabolites and pesticide detoxification [46]. Studies have shown that when Manduca sexta larvae are fed plant material expressing CYP6B46-specific dsRNA, the level of this transcription decreases and larval growth is hindered [47]. In addition, studies have shown that RNAi silences CYP307A1 and blocks molting steroid synthesis, suggesting that this gene is required for molting steroid biosynthesis in anopheles gambiae. Silencing CYP6B7 alone or CYP6B7 in combination with CPR and/or Cyt-b5 increased the sensitivity of bollworm to fenvalerate, indicating that CYP6B7, CPR and cyt-b5 were synergistic in the metabolic enhancement of fenvalerate and played an important role in the resistance of bollworm to fenvalerate [48]. After RNAi silencing of CYP321E1, Plutella xylostella has increased sensitivity to chloroaniline, with a mortality rate of up to 70% 49 (Hu et al., 2014). In our study, RNA interference (RNAi) was used to investigate the function of selected target genes. After injection of dsCYP6AB60 and dsCYP321A19, the tolerance of the 4th instar larva of Spodoptera litura to plant allelochemicals (quercetin, coumarin, soy isoflavones) was significantly reduced.

Conclusion
In our study, some P450 genes of S. litura fed on different host plants and artificial diets with tannin were up-regulated compared with those fed on artificial diets, but all glutathione S-transferase genes were down-regulated. The bioassay data showed that tannin can inhibit the growth of S. litura, which indicated that when S. litura faced harmful allelochemicals, its primary detoxification metabolism optoins were narrowed to Cytochrome P450 genes, and the detoxification metabolism of glutathione metabolism was less utilized.
In addition of detoxification metabolism, digestion was another important function in S. litura. In this study, the expression of digestive enzymes genes was assessed, and we found that, in general, the expression of genes encoding proteinases, lipases, and carbohydrases corresponded to food nutritional composition.

Insect Rearing
Insects were purchased from Henan Jiyuan Baiyun Industry Co., Ltd, and then S. litura were fed on artificial diet: 16.7 g of agar, 100 g of soybean flour, 100 g of wheat germ flour, 100 g of oatmeal, 60 g of yeast powder, 40 g of sucrose, 6 g of ascorbic acid, 2 g of sorbic acid, 2 g of methyl paraben, Beijing huinong fumin technology co. LTD) and cabbage (Jingfeng 1, Fuyichun seed sales co. LTD). All plants were planted in a climate chamber maintained at (26 ± 1) °C, 65 ± 5% humidity, and a 14:10 (Light : Dark) photoperiod. Plants that were 1 month old were used for feeding assays. Additionally, 15 g and 7.5 g tannin were added to artificial diets as treatment 1 (Tannin1) and 2(Tannin2), respectively. After that, three treatments of quercetin, coumarin and soybean isoflavone artificial feed (1 mg/g) were set up respectively to verify the function of P450 gene.
S. litura larvae were reared on an artificial diet until they grew to the third instar (L3), then they were

RNA-seq Data Analysis
Raw reads of fastq format were initially processed using in-house perl scripts. The clean reads were then obtained by removing reads containing adapter, poly-N, and low quality reads from raw reads.
The Q20, Q30, GC-content, and sequence duplication level of the clean reads were then calculated.
These clean reads were then mapped to the reference genome sequence using hisat2 tools software.
The databases of Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO, and GO were used for gene function annotation. Gene expression levels were estimated by fragments per kilobase of transcript per million fragments mapped. Differential expression analysis of the two groups was performed using the DESeq R package (ver. 1.10.1). The resulting P values were adjusted using Benjamini and Hochberg's approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 found by DESeq were assigned as differentially expressed. Gene Ontology (GO) enrichment analysis of the differentially expressed genes (DEGs) was implemented by the GOseq R package based on Wallenius non-central hyper-geometric distribution [50]. KOBAS [51] software was used to test the statistical enrichment of differentially expressed genes in KEGG pathways.

Quantitative Real-time PCR Validation
Total RNA was extracted as described above and Reverse Transcription System (Takara) was used for cDNA synthesis. The relative expression levels of P450, UDP-glucosyltransferase and GST were conducted by quantitative real-time PCR (qRT-PCR). dsRNA of the GFP gene used as a negative control was synthesized by the same method as above, and primers used were listed (Table S1). The obtained dsRNA was detected by ultraviolet spectrophotometry and the purity and integrity were evaluated by agarose gel electrophoresis. The final concentrations of dsRNA were adjusted to 1.5 µg·µL − 1 by RNase-free water and kept at -80 °C.
For RNAi bioassays, the final concentrations of dsRNA were adjusted to 1.5 µg/µL using DEPC-treated (RNase-free) water prior to use. All dsRNA injection experiments used 4th instar larvae (day 1 and hunger for 4 h) of S. litura, with 2µL (3.0 µg) of dsRNA injected into the distal second segment of the abdomen by manual microinjector, while the control group was injured with an equivalent volume of dsGFP. The treated larvae were fed with artificial feed supplemented with plant allelochemicals (The larvae injected with dsCYP321A19 were fed with quercetin, and the larvae injected with dsCYP6AB60 were fed with coumarin and soybean isoflavones).
For the RNAi efficiency evaluation, the midguts and fat bodies of the surviving larvae (6 larvae (9)

Table S1
Primers used in this study. Figure 1 The weight of S. litura larvae fed on different diets. The data are mean ± SD and different letters indicate significant difference at the 0.05 level by the Duncan's multiple range test.

Figures
AD: artificial diet. T: timepoint. were fed an artificial diet. RT-qPCR analysis was used to determine the relative transcript levels for each gene. Data shown as means ± SE derived from three biological replicates.
Different letters above bars indicate significant differences (p < 0.05) according to the Duncan's multiple range test (same as below).

Figure 3
Expression levels of CYP321A19 sequences when exposed to quercetin (A), and CYP6AB60 sequences exposed to coumarin (B), soy isoflavones (C). Data shown as means ± SE derived from three biological replicates (Student's t-test, ** p < 0.01, * p < 0.05, same as below).

Figure 4
Effect of CYP321A19 and CYP6AB60 silencing on S. litura resistance to allelochemicals.
Knockdown reduction rates of CYP321A19 in midgut (A) and fat body (B) after injection of dsRNA. Relative expression of CYP6AB60 in the midgut and fat bodies of larvae exposed to coumarin (C, D) and soy isoflavones (E, F) after dsRNA injection. Control larvae were injected with dsGFP.

Figure 5
Changes in larval body weight of S. litura following smear of dsRNA. After dsCYP321A19injected larva, the net weight increased (A) and the weight increased per day (B) after feeding with quercetin. After dsCYP6AB60-injected larva, the net weight increased (A) and the weight increased per day (B) after feeding with coumarin (C, D) and soy isoflavones (E, F).