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 (T0).
Two days later (T1), 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 (T3), 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.
Treatment | T1 | T2 | T3 |
Cotton | 0.35 ± 0.01 de | 0.23 ± 0.00 a | 0.16 ± 0.00 a |
Cabbage | 0.40 ± 0.00 a | 0.24 ± 0.00 a | 0.16 ± 0.00 a |
Corn | 0.37 ± 0.01 cd | 0.21 ± 0.00 b | 0.15 ± 0.00 a |
Soybean | 0.39 ± 0.01 ab | 0.23 ± 0.00 a | 0.16 ± 0.00 a |
AD | 0.38 ± 0.00 bc | 0.23 ± 0.00 a | 0.16 ± 0.00 a |
Tannin1 | 0.33 ± 0.01 f | 0.17 ± 0.01 d | 0.12 ± 0.00 a |
Tannin2 | 0.35 ± 0.02 e | 0.19 ± 0.01 c | 0.13 ± 0.00 a |
Those in the same column (mean ± SD) followed by different letters show significant difference at the P < 0.05 level by Duncan's multiple range test. AD: artificial diet. T: timepoint. |
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
Table 2 Statistic results of new gene function annotations
Annotated databases | New Gene Number |
COG | 27 |
GO | 91 |
KEGG | 69 |
KOG | 100 |
Pfam | 136 |
Swiss-Prot | 87 |
eggNOG | 223 |
nr | 345 |
All | 349 |
Differentially Expressed Genes Between Treatments
Gene expression was analyzed based on the comparison of results. Differentially expressed genes were identified according to their expression levels in different samples, and functional annotation and enrichment analyses were performed. S. litura midgut transcriptomes were studied by RNA-seq to determine changes in expression level in response to feeding from different diets. Gene expression levels for each replicate were assessed using principal component analysis (PCA), and the results revealed obvious differences from different diets. The gene expression in animals fed on artificial diets that contained 15 g/L and 7.5 g/L tannin clustered together and were far from the other treatments based on sample scores for the first (PC1) principal components. These results indicated that S. litura midgut modified gene expression levels in response to different diets (Supplement Fig. 1A).
The purpose of this study was to identify the key genes of S. litura that enable the insect to adapt to plant secondary metabolite, In order to investigate this, we carried out a pair-wise comparison of S. litura fed on other diets against artificial diets was carried out. Samples fed on cabbage, corn, cotton, soybean, and artificial diet (including tannin) exhibited 1,912 (964 up and 948 down), 1,395 (769 up and 626 down), 2,069 (1025 up and 1044 down), 2,998 (1516 up and 1482 down), and 1533 (838 up and 695 down) differentially expressed genes, respectively. Samples grown on artificial diet contain 15 g/L and 7.5 g/L tannin displayed similar gene expression, so those samples were combined as one sample set (Supplement Fig. 1B).
We next used a venn diagram to analyze the distribution of differentially expressed genes. Only 45 up-regulated and 64 down-regulated differentially expressed genes were involved in all treatments, and there were 238 up-regulated and 244 down-regulated differentially expressed genes when tannin treatments were excluded from venn diagram analysis (Supplement Fig. 1 C-F). The unique differentially expressed gene numbers were higher in cotton and soybean treatment samples, both in up-regulated and down-regulated genes analysis.
Identification Of Putative Pathway Related To Diet Adaption
The differentially expressed genes in pair-wise comparison of S. litura fed on various plant diets against artificial diets were subjected to KEGG pathway database analysis to discovery any significant changes to metabolic pathways genes. In top 5 up-regulated pathways, three enriched pathways (Metabolism of xenobiotics by cytochrome P450, Drug metabolism - cytochrome P450, and Pentose and glucuronate interconversions) were present across four treatments. Two enriched pathways (Glutathione metabolism and Ascorbate and aldarate metabolism) were present across three treatments. In the top 5 down-regulated pathways, one enriched pathway (Neuroactive ligand-receptor interaction) was shared across four treatments, and 5 enriched pathways (Endocytosis, Phosphatidylinositol signaling system, N-Glycan biosynthesis, Alanine, aspartate and glutamate metabolism and other glycan degradation) were shared across the treatments (Supplement 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 (Fig. 3A). Similarly, when coumarin or soy isoflavones were added to the artificial diet, the expression levels of CYP6AB60 increased significantly compared with the control group (Fig. 3B, C).
Functional analysis of CYP321A19 and CYP6AB60 by RNAi
To evaluate the role of CYP6AB60 in the detoxification of plant allelochemicals, CYP321A19 and CYP6AB60 were silenced by RNAi technique in 4 instar larvae. Compared to the control group (injection of dsGFP), transcriptional levels of CYP321A19 were significantly decreased by 83.9% and 66.2% in the midgut and fat body at 72 h (Fig. 4A, B). Similarly, in both midgut and fat body tissues, CYP6AB60 transcript levels were significantly reduced following dsRNA injection (Fig. 4C-F).
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).
Quantitative real-time PCR validation
To verify the transcriptome data, we examined the relative expression levels of P450 (LOC111350062, LOC111358240, LOC111351731), UDP-glucosyltransferase (LOC111348983, LOC111348863, LOC111348860) and GST (LOC111354038, LOC111351682, LOC111352663, LOC111351550). The qRT-PCR of these unigenes showed that the results were consistent with the DGE results (Figure 6).