Background

Most herbivorous insects are restricted to a narrow range of hosts, whereas other insects are adapted to a wider host range [1]. Generally, successful host adaptation involves several essential traits of herbivorous insects, including the abilities to detect the correct plants, acquire nutrients while avoiding intoxication, and overcome plant defenses [2]. Absence of these essential traits have been reported to result in the failure of an insect to feed on specific plants [3,4,5,6]. To date, studies of diet-associated responses have mainly focused on a specific insect fed on different diets [7]. The research findings have shown that the genes associated with detoxification, digestion, and transport are significantly influenced by host transfer [7]. However, knowledge of adaptation to disparate hosts in different insect species is scarce, and most studies have primarily focused on differences in detoxification across species in response to plant secondary metabolites [8,9,10]. Comparative transcriptomic analysis of closely related taxa provides an ideal approach for revealing the distinctions in host adaptation across herbivorous species.

The brown planthopper (Nilaparvata lugens, BPH) and the small brown planthopper (Laodelphax striatellus, SBPH) are closely related insect pests in the family Delphacidae. Although they both use rice plants as their primary food source, their host ranges differ. BPH is a monophagous insect pest restricted to rice plants, whereas SBPH is an oligophagous insect that can feed on rice, wheat, and other gramineous plants [11]. In a wheat-rice rotation system, SBPH, which is able to overwinter in temperate zones, shifts between rice and wheat plants each year [12]. In contrast, the northern border of the overwintering areas of BPH is at approximately 21–25°N. The migratory BPH is seldom in contact with wheat plants in the locations where BPH overwinters [

Results

Performance of planthoppers on rice and wheat plants

Rice plants, but not wheat plants, can be colonized by BPH with only rice-colonizing BPH strains (rBPH) produced under laboratory conditions. In contrast, SBPH can successfully colonize both rice and wheat plants, and two SBPH strains (rSBPH and wSBPH) were maintained in our laboratory for over 30 generations. According to the 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 days) than those provided with water only (LT50 = 3.3 days). These results indicate that BPH could ingest wheat sap and survive on wheat plants for a short time but not for an extended period. For SBPH, both rSBPH and wSBPH successfully survived on rice and wheat plants (Fig. 1b), which is consistent with previous reports [28].

Fig. 1
figure 1

Survival of planthoppers on rice and wheat plants. a The survival of rBPHs colonizing rice plants, transferred to wheat plants, and provided with water only. b The survival of rSBPH colonizing on rice plants and transferred to wheat plants, and wSBPH colonizing wheat plants. Light shades indicate 95% confidence intervals. Different letters signify significant different survival distributions among each treatment group at P < 0.05 according to the log-rank test

Full size image

Overview of RNA sequencing data

To explore the mechanism underlying the different performances of the two planthoppers, rice-colonized planthoppers were transferred to wheat plants for 24 h (short-term transfer) or reared on wheat plants for over 30 generations (long-term colonization). The guts of planthoppers that colonized rice plants (rSBPH and rBPH) or wheat plants (wSBPH) or were transferred from rice plants to wheat plants (tSBPH and tBPH) were then isolated and underwent high-throughput sequencing. A total of 15 libraries (5 treatments and 3 biological replicates for each treatment) were generated, with clean reads exceeding 45 million in each library. The clean reads were mapped to their reference genomes [18, 21]. For BPH, 75–83% of clean reads were mapped to the reference genome. For SBPH, 60–66% of clean reads were mapped to the reference genome. According to the saturation analysis, the number of detected genes decreased as the number of reads increased, and the 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 patterns of wSBPH and tSBPH were closely related, indicating that host plants exerted a nonnegligible influence on gene expression (Fig. S2, Supporting information).

Analysis of differentially expressed genes (DEGs)

Gene expression changes were analyzed by comparing rice colony planthoppers to transfer planthoppers (tSBPH vs rSBPH and tBPH vs rBPH) and rice colony planthoppers to wheat-colony planthoppers (wSBPH vs rSBPH) using a threshold change of > 2-fold and an FDR-adjusted p-value of < 0.05. For rice colony planthoppers transferred to wheat plants, a total of 2877 and 2638 genes were differentially expressed in SBPH and BPH, respectively (Fig. 2). There were 2372 genes upregulated and 505 genes downregulated when rSBPH was transferred to wheat hosts (Table S1). Among these DEGs, genes participating in signal transduction were particularly upregulated. CYP4DE1, which mediates wheat adaptation and ethiprole tolerance [15] in SBPH, was also significantly induced after transfer (Fig. S3, Supporting information). In contrast, 71 genes related to ribosomal proteins and 48 genes related to oxidative phosphorylation were significantly downregulated, indicating decreased protein production and energy metabolism (Fig. S4, Supporting information). In BPH, the number of genes downregulated (2171 genes) exceeded the number of genes upregulated (467 genes) (Table S2). The majority of genes that were significantly downregulated were associated with intestinal mucins, serine proteinases, and sugar transporters. In addition, reduced expression was also found for detoxification-related genes (Fig. S3, Supporting information), which included 9 ABC transporters, 8 P450s, 5 UGTs, and 1 GST. In contrast to SBPH, the majority of ribosomal proteins were upregulated in BPH (Fig. S4, Supporting information). Cuticular proteins, which form the insect cuticle and are involved in insect molting, were dramatically upregulated after BPH was transferred to wheat (Table S2).

Fig. 2
figure 2

The number of differentially expressed genes in planthoppers that feed on different hosts. The differentially expressed genes were analyzed by comparing tSBPH to rSBPH, wSBPH to rSBPH, and tBPH to rBPH based on a threshold of > 2-fold change and an FDR-adjusted p-value of < 0.05

Full size image

In the comparison of rSBPH and wSBPH, a total of 2516 DEGs were identified (Fig. 2). Strikingly, 90.9% of DEGs (2288 genes) showed higher expression in wSBPH than in rSBPH (Table S3), with genes related to peroxisomal biogenesis factor, nucleotide exchange factor, peptide transporter, and CYP6FK1 exhibiting the most dramatic changes. Similar to the patterns of rSBPH transferred to wheat hosts, 37 genes participating in signal transduction were significantly enriched. Among the 228 downregulated genes, the most dramatic changes in zinc metalloproteinase, UGT, and alpha-glucosidase were observed. Other downregulated genes participating in chitin metabolism, carbohydrate derivative metabolism, starch and sucrose metabolism, and oxidative phosphorylation were significantly enriched (Table S3).

Classification of SBPH genes associated with diet changes

To elucidate the successful colonization of SBPH on rice and wheat plants, the DEGs of SBPH for 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, the DEGs of SBPH were classified into four types (Fig. 3): I) genes changed in the same direction for short-term transfer and long-term colonization, II) genes changed in opposite direction for short-term transfer and long-term colonization, III) genes changed in response to short-term transfer but not long-term colonization, and IV) genes changed in response to long-term colonization but not short-term transfer.

Fig. 3
figure 3

Classification of DEGs in SBPH based on their expression patterns. The expression patterns of identified DEGs in SBPH can be classified into four types: Type I, expression changed in the same direction for short-term transfer and long-term colonization; Type II, expression changed in the opposite direction for short-term transfer and long-term colonization; Type III, expression changed in the short-term transfer, but not in the long-term colonization; Type IV, expression changed in the long-term colonization, but not in the short-term transfer. The number of genes belonging to each type is listed following the heat map. The red and green boxes represent up- and down-regulated genes, respectively, in tSBPH and wSBPH relative to those of rSBPH. The white boxes represent genes that did not change

Full size image

There were 1558 genes associated with a type I response (Fig. 3; Table S4). The enrichment analysis showed that genes participating in signal transduction and immune system were significantly overrepresented. Only 22 genes were associated with a type II response (Fig. 3; Table S4). Four genes were downregulated after transfer but were dramatically upregulated during colonization, whereas the other 18 genes showed the reciprocal expression pattern. There were 1297 genes associated with a type III response (Fig. 3; Table S4). The enrichment analysis showed that the 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 colonized wheat plants over the long term. A total of 936 genes were associated with a type IV response (Fig. 3; Table S4). However, 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 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 1995 gene families were differentially expressed in at least one planthopper species after transfer (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 in both species, 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 these, 293 genes were upregulated in SBPH after transfer, but downregulated in BPH; 55 genes, including 25 ribosomal proteins, were downregulated in SBPH after transfer but were upregulated in BPH.

qPCR validation

To confirm the validity of the 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 a concordant direction of change for the qPCR and transcriptomic results, indicating acceptable accuracy of the DEG transcriptomic results. The heat-shock proteins were significantly upregulated after BPH and SBPH transfer to wheat. The expression levels of the ABC transporters and cytochrome P450 were significantly increased after SBPH transfer to wheat but were significantly decreased in BPH under the same conditions. The ribosomal proteins were significantly downregulated in SBPH after transfer to wheat but were significantly upregulated in BPH after transfer. It is worth noting that two trehalose transporters in BPH showed significantly different changes in expression. The trehalose transporter NLU013658.1 was dramatically downregulated after BPH was transferred to wheat, but NLU003716.1 was dramatically upregulated under the same conditions. In SBPH, the nucleotide exchange factor, peroxisomal biogenesis factor, and peptide transporter were significantly upregulated after transfer, but the venom serine carboxypeptidase-like and maltase were significantly downregulated. In BPH, genes such as cryptosporidial mucin, serine proteinase stubble, and peptide methionine sulfoxide reductase were significantly downregulated after transfer, but the cuticle protein 16.5-like, chemosensory protein, and lipid storage droplets surface-binding protein were significantly upregulated.

Fig. 4
figure 4

Correlation between transcriptomic data and qPCR results in SBPH. The relative expression level of each gene was determined by qPCR (blue) and was compared with the expression of the transcriptomic data (green). a UDP-glucuronosyltransferase, b venom serine carboxypeptidase-like, c sugar transporter, d maltase 2-like, e 60S ribosomal protein, f 40S ribosomal protein, g nucleotide exchange factor SIL1 I, h peroxisomal biogenesis factor 3-like, i 70 kDa heat shock protein, j peptide transporter family 2-like, k cytochrome P450, l ABC transporter of evm. TU.Contig86.54, m ABC transporter of evm. TU.Contig58.174

Full size image
Fig. 5
figure 5

Correlation between transcriptomic data and qPCR results in BPH. The relative expression level of each gene was determined by qPCR (blue) and was compared with the expression of the transcriptomic data (green). a cryptopsoridial mucin, b facilitated trehalose transporter Tret1-like of NLU013658.1, c serine proteinase stubble, d peptide methionine sulfoxide reductase, e elongation of very long chain fatty acids protein, f ABC transporter of NLU003498.1, g ABC transporter of NLU013034.1, h cytochrome P450 6A20, i cytochrome P450 4C3, j cuticle protein 16.5-like I, k facilitated trehalose transporter Tret1-like of NLU003716.1, l chemosensory protein 12, m lipid storage droplets surface-binding protein, n small heat shock protein, o 60S ribosomal protein, p 40S ribosomal protein, q heat shock protein

Full size image

Discussion

The wSBPH and rSBPH used in this study were reared under laboratory conditions for more than 30 generations, whereas the tSBPH was collected by transferring rSBPH to wheat plants within 24 h. Surprisingly, the expression pattern of tSBPH was more similar to wSBPH than rSBPH (Fig. S2, Supporting information), indicating a rapid response by SBPH to the change in host plant and a leading role of the host plant in sha** gene expression. Additionally, the transfer of rSBPH to wheat plants gave rise to broad transcriptional readjustments, and the majority of DEGs were changed in the same direction in response to short-term transfer and long-term colonization. SBPH shifts between rice and wheat plants each year, whereas BPH seldom comes in contact with wheat plants [12, 4, Supporting information). Most genes associated with standard roles in protein translation and ribosomal proteins are generally deemed to be stably expressed and are used as housekee** genes [34]. However, recent studies have demonstrated that ribosomal proteins were differentially expressed in response to host transfers in Bemisia tabaci [35], Helicoverpa armigera [36], Polygonia c-album [37], and Cryptolaemus montrouzieri [38]. In this study, the expression level of ribosomal proteins was significantly downregulated when rSBPH was transferred to wheat but upregulated in rBPH under the same conditions. In an insect-plant model, regulation of ribosomal proteins is believed to counteract the ribosome inactivating proteins (RIPs), which are produced by host plants and inhibit protein synthesis in insects [39]. Different expression patterns of ribosomal proteins in the two planthoppers might influence the ability of the insect to counteract plant RIPs; this deserves further investigation. Additionally, ribosomal proteins play fundamental roles in the cellular process of translation [34]. The large-scale change of ribosomal proteins inevitably influences the regulation of other genes, which might partially explain the distinct variation in gene expression between the two planthopper species.

Genes associated with the amide/peptide biosynthetic process, amino sugar metabolic process, and aminoglycan metabolic process were significantly enriched in both SBPH and BPH after host transfer. Planthoppers are piercing-sucking herbivores that depend on phloem sap containing high concentrations of sugars, amino acids and inorganic ions as their food sources [15, 37]. Previous work has demonstrated that the sucrose concentration of rice phloem sap and wheat phloem sap is very similar, but the latter has a higher concentration of total amino acids compared with the former [19, 20]. In addition, the predominance of amides between the two phloem saps was also very different [19, 20]. In previous work, we found that the BPH genome lacks the ability to carry out de novo synthesis of some essential amino acids, requiring an additional supply from endosymbionts or plant phloem sap [5, Supporting information). Heat-shock proteins are well-known stress proteins that respond to an array of stresses including thermal hardening, oxidative hardening, chemical pesticides, and desiccation [51, 52]. The induced expression of these proteins as a significant molecular chaperone prevents the irreversible denaturation of proteins and enhances the co** capacity of insects in the face of stress [51]. Previously, the influence of diet quality on heat-shock proteins expression was described in Drosophila melanogaster, and diet-induced heat-shock proteins increased the heat and desiccation tolerance of flies [53]. Feeding on wheat was a biotic stressor for BPH and SBPH [16], which might result in increased heat-shock protein expression.

Conclusions

Overall, SBPH successfully coped with wheat hosts, and the majority of DEGs responded similarly to short-term transfer and long-term colonization. Compared with SBPH, BPH showed distinct gene expression changes after transfer to the wheat plants. The different pattern of changes in gene expression between BPH and SBPH might account for their adaptive differences on wheat plants. Specifically, genes associated with sugar transporters and heat-shock proteins showed similar expression trends for BPH and SBPH. Other genes associated with detoxification, ribosomal proteins, and amino acid metabolism were regulated differently between the two planthoppers. Our work increases our knowledge of planthoppers adapting to rice and wheat hosts, and might be useful in pest management.

Methods

Insect strains

The SBPH and BPH populations used in this study were originally collected from a rice field at Huajiachi Campus, Zhejiang University, Hangzhou, China. This rice field (30.271 ° N, 120.199 ° E) was specifically used for field experiments. The rSBPH and wSBPH were generated by rearing SBPHs on fresh rice (** genes (β-actin and GAPDH) as internal controls. qPCR was performed using the ABI 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA) and the SYBR Green Supermix Kit (Yeasen, Shanghai, China). The program was run under the following conditions: denaturation at 95 °C for 5 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. qPCR result was calculated according to a relative quantitative method (2-∆∆Ct) [59]. Three independent biological replicates were performed.