Molecular Characterization and Expression Profiling of Chemosensory Proteins in Male Eogystia hippophaecolus (Lepidoptera: Cossidae)
Eogystia hippophaecolus Hua, Chou, Fang et Chen (Lepidoptera: Cossidae) is a notorious carpenterworm pest of sea buckthorn, Hippophae rhamnoides L. (Elaeagnaceae). Chemosensory proteins (CSPs) are thought to be responsible for initial biochemical recognition during olfactory perception by the insect. We examined the structure, function, and expression profiles of these proteins in four structures (e.g., antennae, labipalp, legs, and external genitalia) of male adults. Molecular weight, isoelectric point, hydrophilicity and hydrophobicity of proteins, and signal peptide prediction of 18 E. hippophaecolus CSPs (EhipCSPs) were investigated via software. Expression profiles in the four male structures were analyzed by fluorescence quantitative real-time polymerase chain reaction. Bioinformatics analysis showed that most EhipCSPs are low-molecular-weight proteins with hydrophobic regions and a high proportion of alpha-helices, consistent with the general characteristics of insect CSPs. Eight EhipCSPs (EhipCSP2, EhipCSP5, EhipCSP7, and EhipCSP13–17) were predominantly expressed in the labipalp (P < 0.01), and three (EhipCSP6, EhipCSP8, and EhipCSP9) were predominantly expressed in legs (P < 0.01). We speculate that these proteins may be related to contact sensations, host recognition, and other functions. Two EhipCSPs (EhipCSP4 and EhipCSP11) were highly expressed in the external genitalia (P < 0.01), suggesting that they may be involved in spousal positioning or mating activities. Most EhipCSPs were differentially expressed in the four structures, with wide overall expression, indicating an important role in olfactory recognition in multiple tissues. Our findings establish the foundation for further investigation of EhipCSPs and potential development of nonpesticide control measures.Abstract
In insects, chemical communication involves sensing of various semiochemicals, and olfaction plays a vital role in processing environmental chemical signals to guide fundamental behaviors, such as searching for hosts, avoiding predators, feeding, and oviposition (Benton 2009, Renou and Guerrero 2000). From the external environment, lipo-soluble odor molecules enter the water-soluble sensillum lymph, and then reach the dendritic membrane of neurons, where they activate receptors on the dendritic membrane, eventually leading to physiological and behavioral changes (Leal 2013). It is widely believed that two types of olfactory proteins, odorant-binding proteins (OBPs) and chemosensory proteins (CSPs), are involved in initial biochemical recognition (Leal 2013; Niu et al. 2016, Pelosi 2005, Zhou et al. 2019). Insect CSPs are a class of small (13 kDa, 100–115 amino acids), acidic, water-soluble proteins with four conserved cysteine residues forming two disulfide bonds. The most basic function of CSPs is to dissolve and transport various lipophilic ligands and, thereby, identify a large number of nonvolatile semiochemicals in the environment, while performing other functions including regulating growth, development, and circadian rhythms (Pelosi 2005, Pelosi et al. 2018). While OBPs are mainly present in antennae, CSPs are highly expressed in all olfactory organs and broadly expressed in various tissues throughout the insect body. Moreover, CSPs are characterized by highly conserved sequence motifs, including (a) YTTKYDN(V/I)(N/D)(L/V)DEIL at the N-terminus, (b) DGKELKXX(I/L)PDAL in the central region, and (c) KYDP at the C-terminus, and most have hydrophobic binding pockets in the interior of the molecule (Jansen et al. 2007, Lartigue et al. 2002, Mosbah et al. 2003, Pelosi 2005, Pelosi et al. 2018, Picone et al. 2001, Wanner et al. 2004).
Eogystia hippophaecolus Hua, Chou, Fang et Chen (Lepidoptera: Cossidae) is a major destructive carpenterworm pest of sea buckthorn, Hippophae rhamnoides L. (Elaeagnaceae), an important soil- and water-conservation shrub species distributed in northern and western China (Zhou 2002). The biological and ecological characteristics of this species have been investigated, along with the disaster-causing mechanism (Changkuan et al. 2004, Shi-xiang et al. 2005). Sex pheromones in the female E. hippophaecolus have been identified and used to develop specific and efficient artificial sex pheromone traps (Changkuan et al. 2004, Fang et al. 2005, Wang et al. 2014). Additionally, olfactory sensilla have been studied using scanning electron microscopy (Hu et al., 2018a). Based on male and female antennae transcriptome data, Hu et al. (2016, 2018b) examined a series of olfactory-related genes to explore expression and functional characteristics of OBPs (sex pheromone-binding proteins). However, because larvae bore deep into trunks and roots and live a complicated and long life cycle, there are no effective methods for controlling the population density of larvae. In recent years, the olfactory system of insects has been investigated in order to interfere with olfactory recognition and regulate insect pest populations through novel pest control strategies.
In one study, 18 E. hippophaecolus CSP (EhipCSP) genes were identified from male and female E. hippophaecolus antennae transcriptome data, and phylogenetic relationships between EhipCSPs and homologs in other species were explored (Hu et al., 2016). However, the molecular mechanisms of olfactory action remain unclear. Herein, we analyzed the sequence characteristics of these 18 EhipCSP sequences and performed fluorescence quantitative real-time polymerase chain reaction (qPCR) to examine their expression profiles in male tissues from four olfactory structures (e.g., antennae, legs, external genitalia, and labipalp).
Materials and Methods
Insects and tissue collection. Eogystia hippophaecolus were collected from infested H. rhamnoides plants in Baishan Forest Farm (N 32°39′, E 119°42′), Jianping County, Liaoning Province, China, during June 2019. Antennae, labipalp, legs, and external genitalia of males were excised, placed in RNAlater (Ambion, Austin, TX), and stored at –80°C until used.
Total RNA was extracted from male and female tissues using TRIzol reagent (Invitrogen, Carlsbad, CA) and an RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). Total RNA integrity was monitored by 1.2% agarose gels, and RNA quantity was measured using a NanoDrop 8000 instrument (Thermo, Waltham, MA). Total RNA was then employed as a template for first-strand cDNA synthesis using a PrimeScript RT Reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan). All products were stored at –20°C.
Sequence analysis of EhipCSPs. Gene sequences were obtained from published transcriptome sequencing (PRJNA328551) (GenBank accession numbers: KX655936–KX655953). Open reading frames (ORFs) and putative amino acid sequences of the 18 EhipCSPs were determined using the online software ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). Based on the amino acid sequences, the online software ExPASy (http://web.expasy.org/protparam/) was used to predict the molecular weight, isoelectric point (pI), and hydrophilicity of proteins. Hydrophobicity of proteins was analyzed using BioEdit software (Hall 1999), and signal peptide prediction was executed using Signa1P5.0 (http://www.cbs.dtu.dk/services/SignalP/). The phylogenetic tree analysis of EhipCSPs with similar CSPs to other insect species were constructed by MEGA 10 software.
Fluorescence qPCR. Primers used for qPCR were designed using online software Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/) (Table 1). The Eogystia hippophaecolus β-actin gene served as an internal reference (Hu et al., 2016).
qPCR was performed on a Bio-Rad CFX96 PCR System (Hercules, CA) in 12.5-µl reactions containing 6.25 µl of SYBR Premix Ex Taq II (No. RR820A; TaKaRa), 1 µl of each primer (10 mM), 1 µl of sample cDNA (2.5 ng of RNA), and 4.25 µl of ddH2O (sterile distilled water). Thermal cycling was performed at 95°C for 3 min, followed by 40 cycles at 95°C for 10 s and 59°C for 30 s, and melting curve analysis at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. Each qPCR experiment was conducted in triplicate with three biological replicates for each transcript. One biological replicate takes approximately 10 insects. Two negative controls lacking cDNA template were included for each reaction. Bio-Rad CFX Manager software (Bio-Rad) was used to normalize expression based on ΔΔCq values versus control samples using the 2–ΔΔCT method (Livak and Schmittgen 2001).
Statistical analysis. Relative expression levels were subjected to one-way analysis of variance followed by Tukey's honestly significant difference tests implemented in SPSS Statistics 25.0 (IBM, Armonk, NY). Values are presented as means ± standard errors (SE).
Results
Analysis of EhipCSP sequences. The molecular characteristics of EhpiCSPs are displayed in Table 2. None of the EhipCSPs include a complete ORF, and 14 contain signal peptides. EhipCSPs are between 102 and 523 aa in length. Furthermore, the molecular weight of 12 EhipCSPs is ∼14 kDa, and the pI values range from 3.79 to 9.54.
Analysis of the hydrophilicity and hydrophobicity of EhipCSPs. The grand average (GRAVY) of hydrophobicity scores ranged from –1.020 to 0.688, and were negative for 17 of the 18 sequences, suggesting they were hydrophilic. From the hydrophobicity analysis (Fig. 1), we concluded that 12 of the 18 EhipCSPs (EhipCSP3–4, EhipCSP7, EhipCSP10, EhipCSP13–18) possessed an obvious hydrophobic region (regions with a positive GRAVY score) (Table 3).
Citation: Journal of Entomological Science 56, 2; 10.18474/0749-8004-56.2.217
Secondary structure of EhipCSPs. As shown in Table 4, the proportion of α-helices in the secondary structure of EhipCSPs was high (59.3% to 87.8%), while the proportion of β-sheets ranged from 30.7% to 60.0%, and the proportion of random coil structure ranged from 7.8% to 18.0%.
Phylogenetic tree analysis of EhipCSPs. Based on a neighbor-joining tree of CSPs (Fig. 2), we found that EhipCSPs were divided into different groups. EhipCSP1, EhipCSP3, EhipCSP5, EhipCSP9, EhipCSP11, EhipCSP12, EhipCSP16, and EhipCSP17 were monophyletic with the big dipteran (Drosophila melanogaster Meigen) and lepidopteran clade. EhipCSP10 was monophyletic with SinfCSP21. EhipCSP8, EhipCSP15, and EhipCSP18 were monophyletic with CSPs of Bombyx mori Linnaeus and Papilio xuthus Linnaeus. EhipCSP2 and EhipCSP6 were monophyletic with P. xuthus. EhipCSP7 was monophyletic with B. mori. EhipCSP4 and EhipCSP13 were monophyletic with many other lepidopteran clade such as B. mori, P. xuthus, Agrotis ipsilon (Rottemberg), and Sesamia inferens Walker.
Citation: Journal of Entomological Science 56, 2; 10.18474/0749-8004-56.2.217
Tissue-dependent distributions of EhipCSPs. We investigated the expression patterns of the 18 EhipCSPs in chemosensory tissues from antennae, legs, external genitalia, and labipalp in males using qPCR (Fig. 3). The results suggest that two EhipCSPs (EhipCSP12 and EhipCSP18) were expressed mostly in antennae (P < 0.01). Three EhipCSPs (EhipCSP6, EhipCSP8, and EhipCSP9) were predominantly expressed in legs. Expression levels of two EhipCSPs (EhipCSP4 and EhipCSP11) were higher in the external genitalia. Intriguingly, high expression of 8 EhipCSPs (EhipCSP2, EhipCSP5, EhipCSP7, EhipCSP13–17) was observed in the labipalp. Expression of EhipCSP5 was extremely high in labipalp (nearly 4,000 times higher than in controls). Furthermore, EhipCSP1 was expressed at high levels in both legs and labipalp. EhipCSP3 and EhipCSP4 were expressed highly in legs and external genitalia, of which EhipCSP4 was highly expressed in the external genitalia. Similar to EhipCSP5, EhipCSP10 was expressed at significantly higher levels in antennae and legs (Tables 5, 6).
Citation: Journal of Entomological Science 56, 2; 10.18474/0749-8004-56.2.217
Citation: Journal of Entomological Science 56, 2; 10.18474/0749-8004-56.2.217
Discussion
Eogystia hippophaecolus is a destructive pest of sea buckthorn that causes huge ecological and economic damage (Shi-xiang et al. 2005, Zhou 2002). The ability to precisely locate the correct host plant is essential for the survival of phytophagous insects because it mitigates feeding, oviposition, and predator avoidance. Therefore, attractants developed from host volatiles are widely applied to control pest populations by interfering with olfactory recognition. An understanding of the olfactory mechanisms can establish the foundation for the development of new attractants or repellents. CSPs are key proteins that perform crucial functions in insect olfactory recognition (Pelosi et al. 2018). In the present study, we performed molecular characterization of 18 EhipCSPs and found that most of the primary and secondary structures corresponded to the general characteristics of insect CSPs, suggesting that they may perform typical CSP functions (Jansen et al. 2007, Lartigue et al. 2002, Mosbah et al. 2003, Picimbon et al. 2000). The neighbor-joining phylogenetic analysis of EhipCSPs demonstrates that they were significantly divided into different groups, which may suggest EhipCSPs could have various functions.
Knowledge of the expression patterns of CSPs in different insect tissues could help to clarify their physiological functions. Compared with OBPs, which occur primarily in antennae, the distribution of CSPs is ubiquitous (Gong et al. 2007, Gu et al. 2011, Yao et al., 2016). Herein, the distribution of EhipCSPs differed among the four tested tissues of male E. hippophaecolus, consistent with previous research with B. mori (Gong et al. 2007), Spodoptera exigua (Hübner) (Gong et al. 2012), Mamestra brassicae (L.) (Nagnan-Le Meillour et al. 2000), Heliothis armigera (Hübner) (Tian-Tao et al. 2011), and S. inferens (Zhang et al. 2013). This implies that broad expression is a general feature of these proteins, and CSPs may be involved in olfactory functions in diverse tissues (Hu et al. 2018a). Furthermore, analysis of the types and distributions of sensilla in six olfactory tissues in male E. hippophaecolus revealed that tricnoid sensilla, basiconica sensilla, and coeloconica sensilla mainly perform olfactory chemical recognition (Hu et al. 2018b). In the present work, many of the EhipCSPs were expressed at high levels in the labipalp and legs, which mainly contain tricnoid and chaetica sensilla, implying that EhipCSPs may be associated with chaetica sensilla, but this requires further investigations with techniques such as in situ hybridization.
EhipCSPs were expressed predominantly in external genitalia, which may help to solubilize hydrophobic semiochemicals such as pheromone compounds, and assist their controlled release into the environment (Pelosi et al. 2018), thereby contributing to spousal positioning and mating activities. Binding experiments with radioactively labeled pheromone showed that CSPMbraA is localized in both antennae and pheromone gland extracts, and both tissues displayed high affinity for ligands, suggesting that these proteins may be involved in transporting hydrophobic molecules through aqueous media. A similar expression pattern was also reported for S. inferens (Zhang et al. 2013), M. brassicae (Jacquin-Joly et al. 2001), Cnaphalocrocis medinalis Guenée (Liu et al. 2015), and Athetis lepigone (Mschler) (Zhang et al. 2017). We speculate that EhipCSPs in external genitalia may perform a dual role in receiving and emitting the same chemical message.
Furthermore, many EhipCSPs displayed biased expression in legs and labipalp, exemplified by EhipCSP5, which was expressed at levels 4,000 times higher in labipalp than in controls, consistent with research with Choristoneura fumiferana (Clemens) (Wanner et al. 2004) and Heortia vitessoides Moore (Picimbon et al. 2001) showing higher expression in labipalp and legs, suggesting that it may be related to contact sensation and host recognition via a gustatory role. Studies on P. xuthus showed that a large number of CSPs are present in female tarsi, indicating potentially important roles in chemoreception as transporters of ligands in oviposition behavior (Ozaki et al. 2008). However, CSPs are expressed in legs, indicating non-chemosensory functions (Cheng et al. 2015, Cheng et al. 2019, Pelosi 2005, Pelosi et al. 2018, Picone et al. 2001, Qiao et al. 2013). RNA interference–based gene silencing of S. exigua CSPs (SexiCSPs) resulted in high mortality and oviposition inhibition, suggesting that CSPs influence the survival and reproduction of S. exigua (Gong et al. 2012). Also, expression levels of CSP9 in Solenopsis invicta Buren are highest at the end of the third instar, and silencing of this gene by RNA interference affects fatty acid biosynthesis and other metabolic pathways, and prevents cuticle development and ecdysis, suggesting that Si-CSP9 may be involved in the network that contributes to the development of third-instar larvae (Cheng et al. 2015). In a study on the cockroach Periplaneta americana L., expression of a CSP was increased dramatically during limb regeneration, indicating that CSPs may contribute to other physiological processes beyond chemosensory functions (Nomura et al. 1992).
Furthermore, evidence indicates that expression of insect CSPs can exhibit distinct patterns during development. In a study on Clostera restitura (Walker), expression of CresCSP3 reached a peak at 3–4 d after eclosion in males and at 4–5 d in females (Hui et al. 2018). Analysis of SexiCSP expression in larval and pupal stages revealed relatively strong expression in pupae, which suggests a possible chemosensory role in pupae and/or a simple correlation with the onset of development of chemosensory tissues during pupation (Gong et al. 2012). Similar phenomena were also reported for Heliothis virescens Fabricius (Picimbon et al. 2001) and B. mori (Gong et al. 2007) in which expression levels of CSPs varied significantly during different developmental stages. There are also reports that CSPs differentially regulated over insecticide exposure in the study of B. mori (Xuan et al. 2015) and Bemisia tabaci (Gennadius) (Liu et al. 2014, 2016). Those studies led to the hypothesis that CSPs might be involved in physiological processes beyond chemosensory functions, such as host localization, mate allocation, development, and insecticide resistance. In conclusion, our findings provide a foundation for further structural and functional research, could support the screening of potential attractants or repellents, and may assist the development of novel pest control strategies.
Hydrophobicity of 18 Eogystia hippophaecolus chemosensory proteins (EhipCSPs).
The neighbor-joining phylogenetic analysis of chemosensory proteins (CSPs) of Eogystia hippophaecolus (EhipCSP, red) was performed with reference CSPs of Bombyx mori (BmorCSP), Helicoverpa armigera (HarmCSP), Heliothis assulta (HassCSP), Seamia inferens (SinfCSP), Papilio xuthus (PxutCSP), Agrotis ipsilon (AipsCSP), and Drosophila melanogaster (DmelCSP, Diptera). The stability of the nodes was assessed by bootstrap analysis with 1,000 replications, and only bootstrap values ≥0.75 are shown at the corresponding nodes.
Transcript levels of the 18 CSPs in four tissues in male Eogystia hippophaecolus (A indicates antennae; L, legs; G, external genitals; La, labipalp). β-actin served as an internal reference gene for normalizing target gene expression (Hu et al., 2016). Standard errors are represented by error bars, different lowercase letters (a, b, c, d) above bars denote significant differences at P < 0.05, and different capital letters (A, B, C, D) above bars denote significant differences at P < 0.01.
Continued.
Contributor Notes