Performance of planthoppers on rice and wheat plants
Rice plants, but not wheat plants, can be well colonized by BPH, with only rice-colonized BPH strains (rBPH) generated in laboratory conditions. In contrast, SBPH could successfully colonize on both rice and wheat plants, and two SBPH strains (rSBPH and wSBPH) were maintained in our laboratory for over 30 generations. According to survival analysis, more than 90% of rBPH survived on rice plants for 12 days, which was significantly higher than that on wheat plants (Fig. 1A). Additionally, rBPH survived longer on wheat plants (LT50=6.1 day) than that provided with water only (LT50=3.3 day). These results indicated that BPH could uptake wheat sap and survive on wheat plants for a short time, but not for a long time. For SBPH, both rSBPH and wSBPH could survive on rice and wheat plants successfully (Fig. 1B) in accordance with previous reports [30].
Overview of RNA sequencing data
To explore the mechanism underlying different performances of two planthoppers, rice-colonized planthoppers were transferred to wheat plants for 24 hours (short-term transfer), or reared on wheat plants for over 30 generations (long-term colonization). Then, guts of planthoppers that colonized on rice plants (rSBPH and rBPH) or wheat plants (wSBPH), or transferred from rice plants to wheat plants (tSBPH and tBPH) were isolated and underwent Illumina HiSeq2500 sequencing. A total of 15 libraries were generated, with clean reads exceeding 45 million in each library. The clean reads were mapped to their reference genomes [18, 19], respectively. For BPH, there were 75% to 83% clean reads mapped to the reference genome. For SBPH, the percentage ranged from 60% to 66%. According to saturation analysis, the number of detected genes decreased as that of reads increased, and library capacity reached saturation when the number of sequence reads approached 20.0 million (Fig. S1, Supporting information). Furthermore, principal component analysis (PCA) demonstrated that the expression pattern of wSBPH and tSBPH was closely related, indicating that host plants exert a non-negligible influence on gene expression (Fig. S2, Supporting information).
Analysis of differentially expressed genes (DEGs)
Gene expression changes were analyzed via comparing rice-colony planthopper to transfer planthopper (tSBPH_vs_rSBPH and tBPH_vs_rBPH) and rice-colony planthopper to wheat-colony planthopper (wSBPH_vs_rSBPH) using a threshold of >2 fold change and an FDR adjusted p-value <0.05. For rice-colony planthoppers transferring to wheat plants, a total of 2,877 and 2,638 genes were differentially expressed in SBPH and BPH, respectively (Fig. 2). There were 2,372 genes upregulated and 505 genes downregulated when rSBPH was transferred to wheat hosts (Table S1). Among them, genes participating in signal transduction were remarkably upregulated. CYP4DE1, which mediated wheat adaptation and ethiprole tolerance [28] in SBPH was also significantly induced after transferring (Fig. S3, Supporting information). On the contrary, 71 genes related to ribosomal proteins and 48 genes in relation to oxidative phosphorylation were significantly downregulated, indicating a decreased protein production and energy metabolism (Fig. S4, Supporting information). In BPH, the amount of genes downregulated (2,171 genes) exceeded that of genes upregulated (467 genes) (Table S2). The majority of genes associated with intestinal mucins, serine proteinases, and sugar transporters were significantly downregulated. Besides, reduced expression was also found in detoxification-related genes (Fig. S3, Supporting information), which includes 9 ABC transporters, 8 P450s, 5 UGTs, and 1 GST. Contrary to SBPH, the majority of ribosomal proteins were upregulated in BPH (Fig. S4, Supporting information). Cuticular proteins, which formed the insect cuticle and participated in insect molting, were dramatically upregulated after BPH transferred to wheat (Table S2).
In the comparison of rSBPH and wSBPH, a total of 2,516 DEGs were identified (Fig. 2). Strikingly, 90.9% of DEGs (2,288 genes) showed higher expression in wSBPH than those in rSBPH (Table S3), with peroxisomal biogenesis factor, nucleotide exchange factor, peptide transporter, and CYP6FK1 exhibiting most dramatic changes. Similar to the patterns of rSBPH that transferred to wheat hosts, as many as 37 genes participating in signal transduction were significantly enriched. Among the 228 downregulated genes, there were the most dramatic changes in zinc metalloproteinase, UGT, and alpha-glucosidase. Other downregulated genes participating in chitin metabolism, carbohydrate derivative metabolism, starch and sucrose metabolism, oxidative phosphorylation were significantly enriched (Table S3).
Classification of SBPH genes associated with diet changes
To help elucidate the successful colonization of SBPH on rice and wheat plants, the DEGs of SBPH between short-term transfer (rSBPH vs tSBPH) and long-term colonization (rSBPH vs wSBPH) were analyzed. Based on the gene expression changes in response to different diets, DEGs of SBPH were classified into four types (Fig. 3): I) genes changed in the same direction between short-term transfer and long-term colonization, II) genes changed in the opposite direction between short-term transfer and long-term colonization, III) genes changed in short-term transfer, but not long-term colonization, and IV) genes changed in long-term colonization, but not short-term transfer .
The number of type I responsive genes (1,558 genes) ranked the first (Fig. 3; Table S4). Enrichment analysis showed that genes participating in signal transductions and immune systems were significantly overrepresented. Nevertheless, only 22 genes belonged to type II response (Fig. 3; Table S4). Four genes were downregulated after transferring, but dramatically upregulated during colonization, while other 18 genes showed reciprocal expression patterns. There were 1,297 genes belonging to type III response (Fig. 3; Table S4). Enrichment analysis showed that ribosome pathway, oxidative phosphorylation pathway, and retrograde endocannabinoid signaling pathway were significantly overrepresented. It was noteworthy that the expression level of ribosome proteins was initially suppressed, but recovered when SBPH long-term colonized on wheat plants. A total of 936 genes belonged to type IV response (Fig. 3; Table S4). Nevertheless, we failed to find GO terms or KEGG terms that were significantly enriched. Genes such as integrin alpha-PS4-like, integumentary mucin, and proliferation-associated protein showed a higher expression level in wSBPH.
Comparative genomics of genes in response to host transfer
To comprehend the different diet-associated responses across species, 6139 gene families with only one ortholog in BPH and SBPH were selected and compared. A total of 1,995 gene families were differentially expressed in at least one planthopper species after transferring (Table S5), among which 370 genes were responsive to host transfer in both planthoppers. Interestingly, only 22 genes changed (14 genes upregulated and 8 genes downregulated) in the same direction, including heat shock protein, prophenoloxidase activating factor, MAP kinase-interacting serine/threonine-protein kinase, and small nuclear ribonucleoprotein. Nonetheless, other 348 genes showed different expression patterns between BPH and SBPH. Among them, 293 genes were upregulated in SBPH after transferring, but downregulated in BPH; 55 genes, including 25 ribosomal proteins, were downregulated in SBPH after transferring, but upregulated in BPH.
qPCR validation
To confirm the validity of transcriptomic data, 15 SBPH genes and 18 BPH genes were selected for qPCR analysis. Thirteen SBPH genes (Fig. 4) and 17 BPH genes (Fig. 5) showed concordant direction of change between qPCR and transcriptomic results, indicating the accuracy of DGE transcriptomic results. For heat shock proteins, they were significantly upregulated after BPH and SBPH transferring to wheat. For ABC transporters and cytochrome P450s, their expression levels were significantly increased after SBPH transferring to wheat, but were significantly decreased in BPH at the same condition. For ribosomal proteins, they were significantly downregulated after SBPH transferring to wheat, but were significantly upregulated in BPH after transferring. It was worth to mention that two trehalose transporters in BPH showed significantly different expression changes. The trehalose transporter of NLU013658.1 was dramatically downregulated after BPH transferring to wheat, but the NLU003716.1 was dramatically upregulated at the same condition. In SBPH, the nucleotide exchange factor, peroxisomal biogenesis factor, and peptide transporter were significantly upregulated after transferring, but the venom serine carboxypeptidase-like and maltase were significantly downregulated. In BPH, genes such as cryptopsoridial mucin, serine proteinase stubble, peptide methionine sulfoxide reductase were significantly downregulated after transferring, but the cuticle protein 16.5-like, chemosensory protein, and lipid storage droplets surface-binding protein were significantly upregulated.