De novo comparative transcriptome analysis of a rare cicada, with identification of candidate genes related to adaptation to a novel host plant and drier habitats

For herbivorous insects, incorporation of a novel host into the diet and subsequent formation of distinct host associations play an important role in the early step of speciation process [1]. Plants produce a wide range of allelochemicals to defend against herbivore attack while, in turn, insects have evolved different ways of coping with the chemical barriers. Due to the remarkable diversity of plant secondary compounds, an insect is inevitably confronted with different chemical environments when undergoing a host switch [2]. Consequently, host shift is an important driver of phenotypic and genetic changes for herbivorous insects [3]. Although the importance of insect adaptation to host chemistry has long been recognized, our understanding of the genetic basis of adaptation to new hosts is still insufficient [2].

Subpsaltria yangi Chen (Cicadidae: Tibicininae) is an endemic, Chinese cicada species [4–6], and male’s intraspecific sexual mimicry in this species was revealed recently [7]. This rare species had been known to be distributed in the Loess Plateau and adjacent areas in Shaanxi Province and the Helan (hereafter HL) Mountains, Ningxia Hui Nationality Autonomous Region [4, 5, 7]. During the field investigations of S. yangi since 2011, a few more populations were discovered from the Loess Plateau and adjacent areas in Shaanxi, Shanxi and Gansu provinces [8]. We found that most populations of S. yangi occurring in the Loess Plateau and adjacent areas specialize on the sour jujube Ziziphus jujuba Mill. var. spinosa (Bunge) Hu ex H. F. Chow (Rhamnaceae) (Fig. 1a). However, the population occurring in the Helan Mountains, surrounded by deserts or semi-deserts, utilizes Ephedra lepidosperma C. Y. Cheng (Ephedraceae) (Fig. 1b), although sympatric Z. jujuba var. spinosa is available (see Results). Genetic evidence and acoustic analysis of the calling song of males significantly indicate that the HL population is genetically differentiated from other populations of S. yangi, which provide a possible example of incipient speciation in insects [8]. This, coupled with the fact that Z. jujuba var. spinosa are abundant but not utilized as main host in the Helan Mountains, suggests that the population from the Helan Mountains has experienced a ‘host shift’.

Identifying transcriptional changes associated with host shifts is an important step in understanding the molecular mechanisms of herbivorous insects adaptating to their new hosts [2, 9, 10]. Previous studies have demonstrated transcriptional regulations associated with insects feeding on different hosts. For example, research on host shift in a cactophilic fly, Drosophila mojavensis, has identified cytochrome P450s, glutathione S-transferases and UDP-glycosyltransferases as important gene classes likely involved in cactus utilization [11]. Research on host shift in another cactophilic fly, D. mettleri, revealed that host shift induces a number of genes involved in processes relevant to host adaptation, e.g., detoxification, central metabolism and chemosensory pathways [9]. De Panis et al. [12] suggested that transcriptome modulation during host shift is driven by secondary metabolites in the desert cactophilic fly D. buzzatii. Recently, Zhong et al. [10] suggested that transcriptional regulation of genes related to digestion, detoxifcation and ribosome may play an important role in adaptation of the striped stem borer Chilo suppressalis to a new host, the water-oat Zizania latifolia.

Cicadas are root pests feeding on the xylem fluids of their host plants and then emerge from the soil to use the tree canopies for mating, oviposition, and also additional feeding [13, 14]. Populations of S. yangi feeding on different host plants may confront different chemical environments. The plant Z. jujuba var. spinosa is a thorny, deciduous plant widely distributed throughout the tropical and subtropical regions of the world [15]. The major compounds of branches and leaves of Z. jujuba var. spinosa are rutin, catechin, lupeol, quercetin-3-O-β-D-glucoside, zizyberanalic, epiceanothic and betulic acid [16, 17]. In contrast, plants of the genus Ephedra are known to contain six types of alkaloids, i.e., ephedrine, pseudoephedrine, methylephedrine, methylpseudoephedrine, norephedrine and norpseudoephedrine, which have been used in traditional Chinese medicine for at least 5000 years [18]. Adult cicadas feed exclusively on the xylem fluid from branches of their host plants. Cicada nymphs are long lived underground and feed exclusively on the xylem sap from roots of their host plants. The cicada S. yangi may be a serious pest on the medicinal plant E. lepidosperma. Although the chemical composition in E. lepidosperma is not documented detailedly, this plant has been reported containing two types of alkaloids, ephedrine and pseudoephedrine [19]. Previous studies have shown that breeding in an alkaloid-rich host plant Trichocereus terscheckii [20, 21] affects fitness in D. buzzatii, such as decreasing viability, body size and starvation resistance, and extending development [22–25]. Therefore, adaptation to a new host plant such as E. lepidosperma for S. yangi entails the acquisition of mechanisms aimed to face such chemical challenges. This, coupled with geographic variation in host plant specialization, makes the S. yangi system especially amenable for studies on the genetic basis of new host adaptation.

Herbivorous insects show variability in their ability to use plants, which is based on differences in preference, ingestion speed, digestion efficiency, and detoxification [26, 27]. Olfaction in antennae is essential for regulating insect behaviors such as host preference and oviposition site selection [28]. Many olfactory-related proteins are responsible for perceiving plant volatiles and mediating host selection of phytophagous insects [29, 30]. Recently, Qi et al. [28] conducted transcriptome analysis based on RNA-Seq data from antennae of S. yangi to better understand the olfactory mechanisms in S. yangi which has a very narrow host range far fewer than diets of most other cicadas. They identified a number of olfactory genes of S. yangi, which provide direct evidence for future research of the olfactory system of S. yangi at the molecular level and provide information for elucidation of the molecular mechanisms and evolution of chemosensation in sap-sucking insects.

The midgut, the second largest organ in the insect body, actively interacts with the physical environment [31], which is the entry site for harmful bacteria, viruses and toxins, as well as for food and water [32]. The midgut also plays critical roles in digestion and nutrient uptake and, in particular, detoxification and oxidative stress responses. However, our understanding about the mechanisms underlying the relationship between changes in midgut proteins and adaptation of plant-feeding insects to different host plants is still very limited. Comparative studies are needed to elucidate mechanisms underlying the relationship between the changes in midgut proteins and the adaptation of insects to novel hosts. Thus, we compared gene expression in the midgut of different populations of S. yangi respectively feeding on Z. jujuba var. spinosa and E. lepidosperma. Our main goal was to detect differentially expressed genes (DEGs) in response to different host plants and habitats, and to test the hypothesis that the host plant shift in S. yangi is facilitated by differential regulation of genes related to digestion and detoxification. The results may also be helpful to explain the metabolic mechanism in S. yangi to adapt to new habitats and mechanisms of speciation in herbivorous insects.

RNA-Seq is revolutionizing the fields of ecological and evolutionary genomics, and contributing new insights into the responses that organisms deploy in alternative environments (e.g., plant hosts) [12]. This approach has been employed to evaluate the genetic changes, from a broad view and at the level of candidate genes, in response to different host plants in insects [9, 11, 12, 33, 34]. In the present study, the strongest gene expression differences were revealed in the pairwise comparison between ‘HL vs HC’ (1826 DEGs), followed by the comparison between ‘HL vs FX’ (723 DEGs), while the weakest expression differences were found in the comparison between ‘HC vs FX’ (183 DEGs) (Fig. 3a). This is in line with the genetic differentiation among populations of S. yangi based on mitochondrial (COI + COII + Cytb + A6A8) genes, where the mean Fst among the three comparisons, ‘HL vs HC’, ‘HL vs FX’ and ‘HC vs FX’ was 0.0735, 0.0676 and 0.0121, respectively [8]. This suggests that differential gene expression in the midgut among these populations is closely associated with genetic variations of related populations.

The HC and FX populations of S. yangi specialize on Z. jujuba var. spinosa, while the HL population has shifted to chemically divergent E. lepidosperma. The two host plants differ in biochemical composition and, thus, are expected to have a different nutritional value for S. yangi. Our results show that the genes related to digestion were mainly up-regulated in the HL population when compared with the HC and FX populations (Fig. 4). Our results might indicate that the DEGs related to digestion were involved in adaptation to host shift in S. yangi.

Some differentially expressed metabolism-related genes of insects in adapting to different biochemical composition of host plants have been revealed [9, 12, 34]. For example, Chikate et al. [35] chose a set of trypsin and chymotrypsin transcripts and inspected their pattern of expression of the cotton bollworm Helicoverpa armigera in response to host plants with different nutritional qualities. It is suggested that serine proteases are key enzymes allowing this lepidopteran pest to adapt to many different host plants and, therefore, augmenting polyphagy [35]. Celorio-Mancera et al. [33] reached similar conclusions in the butterfly larvae of Polygonia c-album. In our present study, we also reveal that four genes (Cluster-41,210.32990, Cluster-41,210.95944, Cluster-41,210.113279, Cluster-41,210.51587) encoding serine proteases were differentially expressed in the HL population when compared with the HC and FX populations (Fig. 4; Additional file 10: Table S8), which may be involved in host selection and adaptation of S. yangi.

Besides the nutritional requirements, insects should at the same time be able to cope with toxic chemicals from their host plants. When confronted with host shift, insects can successfully survive on their new host plant through enzymes coded by detoxifying genes such as P450s, GSTs, UGTs and CEs [9, 11, 12, 33, 34]. From the midgut of S. yangi, besides P450s, we also found that five UGTs, six CEs and one GST were differentially expressed in the HL population when compared with the HC and FX populations. In addition to detoxifying enzymes, ABC transporters are also involved in detoxification [36], which are membrane-bound proteins involved in the movement of various substrates, including drugs and insecticides, across the lipid membrane [37]. The ABC superfamily is currently divided into eight distinct subfamilies (A–H) [38]. Among which, three subfamilies, ABCB, ABCC and ABCG, are involved in drug resistance [39]. In our study, differentially expressed ABC transporters were also detected, including ABCB8, ABCC1, ABCC4, ABCC5 and ABCG4, and majority of them were up-regulated in the HL population when compared with the HC and FX populations (Fig. 5; Additional file 11: Table S9). This suggests that these genes may play an important role in detoxification during the host plant shift of this cicada species. Besides the abovementioned proteins that are involved in detoxification, alkaline phosphatase is also associated with removal of alkaloids in pests, and high levels of alkaline phosphatase have been implicated in insecticide resistance [40, 41]. In our study, we found that an alkaline phosphatase, tissue-nonspecific isozyme-like gene (Cluster-41,210.64298) was exclusively expressed in the HL population (Fig. 5; Additional file 11: Table S9), indicating this gene may play a significant role in alkaloid resistance when S. yangi feeds on E. lepidosperma.

Reactive oxygen species (ROS) are derived from the metabolism of molecular oxygen [42], such as superoxide radicals (O₂⁻), hydroxyl radical (OH⁻), H₂O₂, and hydroperoxides (ROOH), which are generated by exogenous and endogenous sources [43]. Exogenous sources, for example prooxidant allelochemicals produced by host plants, pose a serious challenge to herbivorous insects during insect–plant interactions. However, some herbivorous insects have evolved a midgut-based defense against the toxic effects of ROS [44, 45]. They can produce many enzymes for detoxifying ROS, including glutathione peroxidase (GPX), catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase [46]. Mittapalli et al. [44] suggested that the induced expression of antioxidant genes, such as phospholipid glutathione peroxidases, catalases and superoxide dismutases in the midgut of a major insect pest of wheat worldwide, the Hessian fly Mayetiola destructor, to be a protective response to ROS ingested during feeding or food processing. Zhu et al. [47] also suggested that peroxidase potentially involved in insecticide metabolic resistance in the diamondback moth Plutella xylostella. In our present study, two DEGs encoding peroxidase were identified between the populations of S. yangi utilizing different host plants (Fig. 6; Additional file 12: Table S10), suggesting that they may also play an important role in protective response to ROS ingested during feeding or food processing.

Animals living in desert environments are forced to endure intense heat and drought, and as a result, species living in deserts likely involves a large number of adaptive traits, e.g., the ability to maintain the proper water and solute balance [55, 56]. Aquaporins (AQP), a large protein family facilitate transport of water between cells [57] and maintain water balance in the kidney [58–60]. For example, AQP2 transports water back into the kidney from the collecting duct [61, 62] and plays an essential role in the urinary concentrating mechanism in mammals. Mice lacking AQP2 suffer severe polyuria, with average basal daily urine volumes approximately equivalent to body weight and are totally unable to concentrate urine [63]. Similarly, mice lacking AQP1 and AQP4 also presented defects in water reabsorption [63]. While AQP4 is apparently absent in desert species such as kangaroo rats [64] and camelids [65]. In our study, an aquaporin AQPAe.a gene (Cluster-41,210.118935) and an aquaporin 2-like gene (Cluster-41,210.63368) were up-regulated in the HL population when compared with the HC and FX populations (Fig. 6; Additional file 12: Table S10). This suggests that they may play an important role in response to water deprivation for the HL population which occurs in the desert or semi-desert environments of the Helan Mountains. Regarding solute balance, another gene family, solute carriers (Slc), are responsible for the maintenance of electrolyte levels [66, 67]. Several studies have revealed that some members of solute carriers play a significant role in the osmoregulation in desert animals [56, 68–70]. For example, solute carrier family 2 member 9 (Slc2a9) has been suggested to be undergoing positive selection in kidney transcriptomes in the desert-adapted mouse Peromyscus eremicus [69]. A study of differential gene expression in the cactus mouse P. eremicus revealed that Slc2a1 and Slc8a1 showed responses to acute dehydration [56]. Recently, Giorello et al. [71] considered Slc12a2 may be related to water conservation in desert animals. In our present study, a unigene encoding Slc12a2 was up-regulated in the HL population (Fig. 6; Additional file 12: Table S10), suggesting that it may also be related to water conservation in the desert or semi-desert environments.

Host shift and subsequent adaptation can drive evolution and diversification of plant-feeding insects [9, 72]. In the context of a host switch, transcriptional plasticity in metabolic pathways involved in processing and detoxifying food resources may produce a phenotype that is well matched to the new environment (e.g., the deserts or semi-deserts). Directional selection is expected to further alter the plastic response in the direction of the optimum, resulting in adaptive evolution (known as the Baldwin effect) [73]. In this case, the DEGs detected in the midgut of S. yangi might be initially beneficial in the process of host shift in this species, which may subsequently evolve in a long-term feeding on a new host plant (viz., E. lepidosperma). A previous study indicated that the HL population has evolved into a distinct independent lineage according to the analysis of population differentiation based on both molecular data and males’ calling song structure [8]. The deserts or semi-deserts surrounding the Helan Mountains were previously shown as a major dispersal and climatic barrier for gene flow of related animals [74]. Such barriers appear to have promoted divergence of the HL lineage from other lineages of S. yangi [8]. Separated populations are potentially to become distinct species, as separated populations are seldom to get reunited after long-term chance and selection reciprocity, which may cause such populations to diverge and enhance many traits associated with reproductive isolation [75]. Differences in the patterns of gene expression in pairwise comparisons of ‘HL vs HC’ and ‘HL vs FX’ may be representative of the genetic changes which facilitate the colonization of novel habitats for S. yangi, and help to mitigate the stresses imposed by a novel host containing different xenobiotics.

Differential gene expression could be affected by several factors, such as genetic variations between populations [71], response to different host plants [9, 11, 12], response to abiotic factors (i.e., temperature) [76–78], or response to different microbiota in the alimentary canal [79–82]. Our analysis revealed that a certain number of genes were differentially expressed among populations utilizing different host plants and living in different habitats. Furthermore, the weakest expression differences were found between the populations utilizing the same host plant and occurring in the closest habitats. Given that the HL population is locally specialized on the endemic host plant E. lepidosperma but other populations specialize on Z. jujuba var. spinosa, results of our present study suggest that host shift could, coupled with geographical barriers (i.e., the deserts or semi-deserts surrounding the Helan Mountains) and ecological changes, have acted as effective factors resulting in the population divergence and allopatric speciation in S. yangi. Further study is required to confirm that differential expression of the various DEGs identified in our comparative analysis of cicada transcriptomes is due to the mechanisms of adaptation to the novel host plant and new habitats.

In this study, we investigated the transcriptional changes in the midgut of the cicada Subpsaltria yangi. Our results indicate that the DEGs related to digestion, detoxification, oxidation-reduction, stress response, water-deprivation response and osmoregulation may be important in the adaptation to host plants and habitats by S. yangi. The observed differences in gene expression among S. yangi populations occurring in different habitats and utilizing different host plants may help explain the metabolic mechanism of adaptation in S. yangi populations to a novel host plant and drier habitats, and inform future studies on the molecular mechanisms of population divergence or speciation in herbivorous insects.