Transcriptome Analysis of Dauer Moulting of a Plant Parasitic Nematode, Bursaphelenchus xylophilus Promoted by Pine Volatile β-Pinene

Abstract

Pinewood nematode, Bursaphelenchus xylophilus, a pine-parasitic nematode, poses a serious threat to pine trees globally, causing pine wilt disease. When dispersal-stage juvenile 4 (dauer, J_IV, a durable stage) of B. xylophilus enters the new pine, it transforms into a propagative adult (dauer moulting) and reproduces quickly. Our previous studies have found that pine-volatile β-pinene promotes dauer moulting of B. xylophilus; however, this mechanism is not clear. Here, this study is attempting to unravel the molecular process underlying dauer moulting of B. xylophilus through signal chemical tests and transcriptome analysis. The results showed that β-pinene could promote dauer moulting of B. xylophilus, while other common dauer moulting signals, such as dafachronic acid (DA), part of the TGF/insulin signal pathway, were inoperative. Moreover, the J_IV soaked in 1% β-pinene for only 6 h could transform into adults at a significant rate. Therefore, the transcriptomes of J_IV soaked in 1% β-pinene for 6 h were sequenced. It was found that 15,556 genes were expressed; however, only 156 genes were expressed differentially and enriched in the metabolism of xenobiotics, peroxisome, fatty acid metabolism, and carbon metabolism, indicating that energy metabolism was active at the early stage of dauer moulting. With a stricter parameter, the number of differential genes fell to 19, including 4 sterol hydroxylase, 5 dehydrogenase, 2 glucuronosyltransferase, 5 nuclear-related factor, 1 calcium-binding protein, 1 nitrogen metabolic regulation protein, and 1 cystathionine gamma-lyase. These results indicated that dauer moulting of B. xylophilus into adults might not be regulated by the TGF-β/insulin signal pathway but by another new signal pathway related to the 19 differential genes which need more exploration. Our results contribute to the understanding of the molecular mechanisms behind dauer moulting and may be useful in reducing pine wilt disease by suppressing this moulting to cut the life cycle of B. xylophilus.

1. Introduction

The pine wood nematode (PWN), Bursaphelenchus xylophilus—the causal agent of pine wilt disease—is believed to be the greatest killer to pine forests in its introduced regions, such as Japan, China, Korea, and Europe, and poses a serious threat to economic and ecological benefits globally [1,2,3,4,5].

The life cycle of B. xylophilus includes propagative and dispersal stages [6,7]. Under favourable conditions, B. xylophilus goes through the propagative stage, in which it directly develops (egg, juveniles, including J1–J4) into a reproductive adult, lays eggs, and kills the pines, eventually, thus causing pine wilt disease (PWD). However, when the environment becomes unsuitable, B. xylophilus goes into the dispersal/diapause stage. B. xylophilus has two different dispersal and long-lived stages, including dispersal-stage juvenile 3 (J_III) and dispersal-stage juvenile 4 (dauer, J_IV), for resistance to unsuitable environments with unique body structures [8]. J_III is environmentally induced by deteriorating conditions in the pine tree from L2. Genus Monochamus spp. are major vector beetles for B. xylophilus to spread from wealthy pines to healthy pines [9]. J_IV is induced from J_III through the presence of emerging adult beetles, as it enters the trachea of the beetles [10]. While Monochamus species are feeding on healthy trees, high CO₂ concentration released from the trachea by the high respiration rates of the beetles drives lots of J_IV to leave the beetle and swarm into healthy pines to transform into adults [7,11]. Thus, J_IV transforming into adults (dauer moulting) is a key event leading to B. xylophilus spreading among pines.

In response to severe environments, most nematodes, such as Caenorhabditis elegans, a free-living model nematode, also undergo a dispersal/diapause stage, called dauer arrest, prior to the reproductive stage [12]. The molecular underpinnings of the dauer pathway have been reviewed here [13], but a short summary of the essentials is as follows: When the favourable environmental signals (low population density, nutrient supply, and reproductive temperature) are detected by GPCRs (G-protein-coupled receptors) of C. elegans, the signals will be transduced via G-proteins with the transmembrane protein, DAF-11, which converts GTP to cGMP. For reproduction and development, a high level of cGMP leads to the activation of TGF-β and insulin pathways. Activated receptor DAF-2 finally phosphorylates DAF-16/FOXO. The DAF-1/4 receptor kinases are banded to DAF-7/TGF-β, which brings about the phosphorylation of DAF-8 and DAF-14/ SMADs. Nuclear localization of DAF-8 and DAF-14 SMADs up-regulates the expression of hormone synthesis enzymes (DAF-9/CYP450), which produces dafachronic acid (DA), the ligand for DAF-12/NHR. In target tissues, liganded DAF-12 promotes reproduction and inhibits dauer programs. These studies give us a better understanding of the molecular mechanism of the dauer moulting of C. elegans, which is helpful for the research of dauer moulting of B. xylophilus into adults.

Our previous studies have found that it is not the common dauer moulting factors of nematodes, such as suitable temperatures, nutrient availability, or nutrient density, but pine volatiles—especially β-pinene and β-myrcene—being in contact with J_IV for 2 days that promotes J_IV moulting into adults in B. xylophilus [14]. The transformation process of J_IV has been divided into five stages, including J_IV, the transformation prophase (TP), the transformation metaphase (TM), the transformation anaphase (TA), and the transformation telophase (TT) [15]. However, how the pine volatiles promote J_IV moulting is not clear. Here, the shortest contacting time of β-pinene to J_IV was found—and the transcriptome of J_IV, promoted by β-pinene, was analysed—to find the key genes associated with the dauer moulting of B. xylophilus, which is helpful for exploring the molecular mechanism of J_IV moulting and may contribute to preventing pine wilt disease.

2. Materials and Methods

2.1. B. xylophilus J_IV Collection Using Vector Beetle Monochamus Alternatus

The artificial rearing pupae of Monochamus alternatus were obtained from Liangjian Qu’s lab. Propagative B. xylophilus were grown on cultivated Botrytis cinerea using barley medium (10 g barley with 10 mL water in a 50 mL flask sterilized for 30 min) at 25 °C for 10 days and at 4 °C for several days. A single pupa beetle was placed in the flask after its eclosion for 7 days for the formation and transfer of J_IV. The mature beetle, carrying thousands of J_IV, fed on twigs of Pinus massoniana for 7 days to mimic natural feeding conditions. J_IV nematodes were obtained from mature beetles of M. alternatus. The dissected beetle was soaked in ddH₂O for 2 h. The J_IV nematodes were run out of the trachea of beetles and were washed three times. The collected J_IV were stored at 4 °C for the next study.

2.2. Stimulation of the B. xylophilus J_IV Moulting into Adults with Different Chemicals

The final concentrations of the chemicals were NaHCO₃ 298 mM (isotonic), NaCl 298 mM (isotonic), DA (Sigma, Shanghai, China) 1 μM with 0.1% methanol [16], ketoconazole (Ket, Sigma, Shanghai, China) 1 mM with 0.1% methanol [17], 1% β-pinene (Sigma, Shanghai, China) [14], and 0.1% methanol (control). All of the chemicals were diluted in ddH₂O with 5% Triton X-100. Thirty J_IV nematodes were soaked in the prepared chemicals for 2 days in an incubator (25 °C). The numbers of adults were counted using an optical microscope (CZX51, Olympus, Tokyo, Japan), and the transformation rates of the J_IV were calculated. Ketoconazole is an inhibitor of DAF-9, which inhibits the synthesis of DA [17]. These experiments were repeated three times.

2.3. Stimulation of the B. xylophilus J_IV Moulting into Adults with Different Contacting Time of β-Pinene

Thirty J_IV nematodes were soaked in 1% β-pinene for 0 h for the control and 0.5 h, 1 h, 3 h, 6 h, 12 h, and 24 h at 25 °C with a shaker of 200 rpm [14]. The soaked nematodes were washed 3 times with ddH₂O and incubated at 25 °C in incubators, and the transformation rates of the J_IV were calculated at 24 h, 36 h, 48 h, and 72 h. These experiments were repeated three times.

2.4. RNA Extraction of B. xylophilus

Five thousand J_IV nematodes were soaked in 5% Triton X-100 for the control group or in 1% β-pinene with 5% Triton X-100 for 6 h at 25 °C with a shaker of 200 rpm. The nematodes were collected by centrifuging 3 kg for 2 min after being washed three times with ddH₂O. The collected nematodes were disrupted and homogenized with a grinding machine (Jingxin, China). Total RNA was extracted using the Rneasy Mini Kit (Qiagen, Shanghai, China) according to the manufacturer’s protocol. RNA samples were stored at −80 °C. The RNA concentration was determined using a NanoDrop ND-2000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). The quality of RNA was assessed via agarose gel electrophoresis. The integrity of RNA was verified using an Agilent 2100 BioAnalyzer (Agilent, CA, USA). Each sample had three replicates.

2.5. Library Preparation and Transcriptome Sequencing

A total amount of 3 μg of nematode RNA per sample was collected to construct sequencing libraries using a NEBNext^® UltraTM RNA Library Prep Kit (NEB, Ipswich, UK) for Illumin and following the appropriate instructions. According to the protocol steps, we purified the mRNA using poly-T oligo-attached beads. First-strand cDNA was carried out using divalent cations and synthesized using a random hexamer primer and RNase H. Second-strand cDNA was sequenced using DNA polymerase I and RNase H, 250–300 bp cDNA purified with the AMPure XP system (Beckman Coulter, Brea, CA, USA), and adaptor-ligated cDNA at 37 °C for 15 min followed by 5 min at 95 °C. Then, PCR was performed with DNA polymerase, PCR primers, and index primers. The index-coded samples were evaluated on a cBot cluster generation system using the TruSeq PE Cluster Kit v3-cBot-HS (Illumia), according to the instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform, and 150 bp paired-end reads were generated [18].

2.6. Transcriptome Analysis

Reference genomes of B. xylophilus (Accession: PRJNA381109) were downloaded directly from the NCBI genome website. An index of the reference genome was built, and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5.

FeatureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. The FPKM (fragments per kilobase of exon model per million mapped fragments) of each gene was calculated based on the length of the gene and the reads count mapped to this gene. Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted using the edgeR program package through one normalized scaling factor. A differential expression analysis of two conditions was performed using the R package (3.18.1). The p-values were adjusted using the Benjamini & Hochberg method. A corrected p-value of 0.05 and an absolute fold change of 2.0 were used as the significance thresholds for identifying differentially expressed genes (DEGs) [19,20].

Gene Ontology (GO) enrichment analysis of the DEGs was implemented using the clusterProfiler R package. GO terms with corrected p-values of < 0.05 were considered significantly enriched by DEGs [21]. The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a database resource for understanding high-level functions and utilities of the biological system (http://www.genome.jp/kegg/ (accessed on 1 August 2019). The clusterProfiler R package was used to test the statistical enrichment of DEGs of J_IV moulting in KEGG pathways [22].

2.7. Statistical Analysis

In all experiments, the homogeneity of group variances was screened for using Levene’s test. The transformation rates of B. xylophilus J_IV, promoted by different chemicals and different contacting times of 1% β-pinene, were evaluated using one-way ANOVA (analysis of variance) analyses. Data were analysed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). The quantitative data were represented as means ± SE (standard error) [23].

3. Results

3.1. Stimulation of Different Chemicals to the B. xylophilus J_IV Moulting into Adults

It was reported that CO₂ has the function of attracting J_IV dropping out from the trachea of vector beetles [11]. Here, isotonic NaHCO₃ was used to provide CO₂ in soaking buffers and isotonic NaCl and 5% Triton X-100 for the control. The results indicated that there was no significant difference in the transformation rate of J_IV into adults between the stimulation from NaHCO₃ and the control. Meanwhile, there also was no significance in the transformation rate between that of DA, ket, 0.1% methanol, and the control (Figure 1). However, 1% β-pinene showed significant promotion of the transformation of J_IV, similar to our previous study [14].

3.2. Stimulation of β-Pinene with Different Contacting Times to the B. xylophilus J_IV Moulting into Adults

To confirm the action time of β-pinene to J_IV to activate its transformation, the J_IV nematodes were soaked in 1% β-pinene for different amounts of time and transferred into water for the monitoring of their morphological development at different times. The results showed that, at the same detecting times, the transformation rate of J_IV increased along with the time. Even for the 0 h contracting time for the control, 14.76% and 19.37% of J_IV nematodes had transformed into adults at monitoring times of 48 h and 72 h. Along with the increase in the contacting time of β-pinene, the transformation rate of J_IV increased gradually. At the monitoring times of 48 h and 72 h, there were significant differences in the J_IV moulting rates among different 1% β-pinene contacting times. The J_IV transformation rates with β-pinene contacting times of 6 h, 12 h, and 24 h were significantly higher than those of 0 h, 0.5 h, 1 h, and 3 h (Figure 2). The J_IV soaked in 1% β-pinene for at least 6 h could significantly transform into adults, with 59.47% and 71.79% transformation rates at monitoring times of 48 h and 72 h, separately.

3.3. Transcriptome Analysis of J_IV in Contact with 1% β-Pinene for 6 h

To confirm the transformation activation mechanism of J_IV promoted by 1% β-pinene, the transcriptome of J_IV in contact with 1% β-pinene for 6 h was analysed. According to the reference genome of B. xylophilus (Accession: PRJNA381109), 15,556 genes were expressed. However, only 156 genes were differentially expressed between the control groups and β-pinene contact groups (|log2 Fold Change| > 0, p-value < 0.05). After contact with 1% β-pinene for 6 h, J_IV nematodes up-regulated 85 genes and down-regulated 71 genes (Figure 3).

To further explore the potential functions of the identified differentially expressed genes (DEGs) and the pathways involved, GO enrichment and KEGG analyses were performed for the DEGs. According to the GO analysis, 13 DEGs were enriched in biological processes, including catalytic activity, oxidoreductase, and metabolic processes, etc (Figure 4). These genes were annotated to dehydrogenase (8), UDP-glucuronosyltransferase (2), endoglucanase, lipase, and nuclear receptors, with 9 up-regulated and 4 down-regulated. According to the KEGG analysis, DEGs were enriched in the metabolism of xenobiotics by cytochrome P450, peroxisome, fatty acid metabolism, and carbon metabolism, etc. There were 13 genes that were connected together and annotated to glutathione S-transferase-1 (2), UDP-glucuronosyltransferase, thiolase domain-containing protein, thiamine pyrophosphate enzyme domain-containing protein, peroxisomal acyl-coenzyme A oxidase (2), leucyl aminopeptidase, alcohol dehydrogenase, hydrolase, phytanoyl-CoA dioxygenase, fatty acid synthase, and peroxisome, with 2 up-regulated and 11 down-regulated (Figure 5).

Although there were 156 DEGs between the control groups and the β-pinene contact groups, the stricter parameter (|log2 fold change| > 2, p-value < 0.05) and better repeatability were set to find key genes for activating J_IV moulting. Of the J_IV contacted with 1% β-pinene for 6 h, there were 19 genes that were significantly up-regulated, including sterol hydroxylase (4), dehydrogenase (5), glucuronosyltransferase (2), nuclear-related factor (5), calcium-binding protein, nitrogen metabolic regulation protein, and cystathionine gamma-lyase (

Table 1. Nineteen genes up-regulated significantly in dauer juveniles (J_IV) of Bursaphelenchus xylophilus promoted by β-pinene. KEGG was used for analyzing the pathways of genes and GO for analyzing function enrichment.

Classify	Gene Name	Annotation	GO	KO	log2 Fold Change
Sterol hydroxylase	451.9_1968	Protein CYP-33C2	-	K17955	2.522103
	624.9_2079	Cytochrome P450-33C9	GO:0005506;GO:0016705;GO:0020037;GO:0055114	K17955	2.455736
	662.1_1909	Protein CYP-33C2	GO:0016705;GO:0055114;GO:0005506;GO:0020037	K17955	2.429878
	20.7_5387	CYP4BJ1	GO:0005506;GO:0016705;GO:0020037;GO:0055114	K07427	3.887855
Dehydrogenase	225.7_875	Alcohol dehydrogenase	-	-	3.940205
	0.2_1051	Short-chain dehydrogenase	GO:0016491	-	3.67873
	1742.6_1002	Tropinone reductase 2	-	-	3.715131
	215.6_972	Tropinone reductase 2	-	-	2.444587
	58.7_1309	Tropinone reductase 2	GO:0016491	-	2.031488
Glucuronosyltransferase	197.8_2331	UDP-glucuronosyltransferase 2C1	GO:0008152;GO:0016758	K00699	2.180414
	1517.7_779	UDP-glucuronosyltransferase	GO:0008152;GO:0016758	-	2.062236
Nuclear-related factor	166.8_810	Nuclear transport factor 2	GO:0005622;GO:0006810	-	5.771244
	168_711	Nuclear transport factor 2	GO:0005622;GO:0006810	-	2.039707
	202.2_2359	PKc-like superfamily	-	-	3.807015
	25.3_1501	PKc-like superfamily	-	-	3.423471
	487.5_1299	PKc-like superfamily	-	-	2.661725
Calcium-binding protein	693.5_2166	Isochorismatase	GO:0003824;GO:0008152;GO:0005509	-	4.146147
Nitrogen metabolic regulation protein	86.3_630	Nitrogen metabolic regulation protein	-	-	4.204984
Cystathionine gamma-lyase	964.8_3401	Cystathionine gamma-lyase 2	GO:0030170;GO:0003824	K01758	3.295417

). Four sterol hydroxylases were 2 CYP-33C2, P450-33C9, and CYP4BJ1. Five dehydrogenases were 3 tropinone reductase 2, 1 alcohol dehydrogenase, and 1 short-chain dehydrogenase. Nuclear-related factors included 2 nuclear transport factor 2 and 3 PKc, such as superfamily genes.

4. Discussion

Dauer moulting (from dispersal-stage juvenile 4 into adults) is essential for nematodes to complete their life cycles [7,24,25]. Dafachronic acid (DA) is necessary for nematodes to stop diapause and transform into adults, and it is produced by DAF-9 as the ligand of DAF-12 for activating the downstream genes for reproduction and inhibiting dauer programs [13,16,26]. Ketoconazole is an inhibitor of DAF-9, which inhibits the synthesis of DA [17]. Previous studies showed that exogenous DA could promote larval exsheathment and the development of Haemonchus contortus and C. elegans by modulating the dauer-like signalling pathway and lipid metabolism [16,27]. While ketoconazole could completely inhibit dauer recovery, this inhibition could be overcome through pharmacological supplementation of DA [17]. However, in B. xylophilus, DA or ketoconazole had no function on the transformation of J_IV into adults (Figure 1). Moreover, high CO₂ concentration might be associated with J_IV moulting, as it is released from the trachea by the high respiration rates of beetles and drives J_IV nematodes to leave the trachea [11]. So, NaHCO₃ was used to produce CO₂ in the liquid of the J_IV moulting system. However, NaHCO₃ could not promote J_IV transformation into adults in B. xylophilus yet. J_IV moulting of B. xylophilus into adults is a key event in completing its life cycle and leads to PWD spread among healthy pines [7]. Our previous studies showed that β-pinene promoted J_IV moulting into adults in B. xylophilus rather than the common dauer moulting factors of nematodes, such as suitable temperatures, nutrient availability, or nutrient density [14]. In this study, pine volatile β-pinene significantly promoted the transformation of nematodes from J_IV into adults in 2 days. Meanwhile, pine volatiles could attract J_IV taking advantage of these volatile accumulations for its life cycle [14]. Without terpenes, some nematodes of J_IV were also moulted into adults, with transformation rates of 14.76% and 19.37% at monitoring times of 48 h and 72 h (Figure 2). This might be induced by the pine volatiles during the feeding times of beetles.

To know the shortest contact time of β-pinene with J_IV necessary to promote nematode moulting, the J_IV nematodes were soaked in 1% β-pinene for different amounts of time and transferred to water for the monitoring of their morphological development at different times. From the results, the J_IV soaked in 1% β-pinene for at least 6 h could significantly transform into adults (Figure 2). This means that though the chemical signal, β-pinene, was removed after at least 6 h of contact with J_IV, the nematodes could initiate downstream developmental genes for transformation. With stricter parameters (|log2 fold change| > 2, p-value < 0.05), of the J_IV in contact with 1% β-pinene for 6 h, 19 genes were found to be significantly up-regulated (Table 1). In C. elegans, the gene expression microarray was analysed and associated with the dauer recovery (dauer moulting) from the dauer state to the non-dauer state of 0, 1.5, 2, 3, 4, 5, 6, 7, 8, 10, and 12 h. At the transient stage (<3 h), cytochrome P450 enzymes, UDP-glucuronosyltransferases, transporters, and nuclear hormone receptors, such as daf-16, were over-represented. Then, the proteases, acid phosphatases, and ABC transporters involved in long-chain fatty acid transport and digestion were up-regulated. At last, the genes associated with glycolytic, tricarboxylic acid cycle, fatty acid oxidation, and collagens were up-regulated for cuticle and tissue development [28]. Similar to the dauer of C. elegans, the J_IV does not feed during the dispersal stage without stylet but has a mass of neutral lipids in its body [7,29,30]. These lipids are degraded for energy supply and histogenesis for the J_IV moulting into an adult. Overall, 4 sterol hydroxylases and 5 dehydrogenases were associated with fatty acid metabolism, 1 nitrogen metabolic regulation protein was associated with nitrogen metabolism, and 1 cystathionine gamma-lyase was associated with amino acid metabolism. These genes might be related to the J_IV moulting for energy supply and histogenesis. Moreover, 1 calcium binding protein was identified as related to J_IV moulting early signal response. Calcium-binding protein is involved in second-messenger generation for a wide variety of biological processes and proteins that regulate cytoskeletal elements [31]. In response to terpene, a second messenger might be needed for signal transmission of the J_IV moulting process. Nuclear transport factor 2 is identified as stimulating the import of proteins into the nucleus [32]. Here, 2 nuclear transport factor 2 were identified, which might participate in the signal transport from the cytoplasm to the nucleus associated with the J_IV moulting. PKc-like superfamily genes were first defined as histone kinase activity. In C. elegans, PKc family gene TPA1A is related to growth, and PKC1B is required for post-embryonic development of the neurosensory system [33]. Three PKc family genes were identified that might participate in the histogenesis for J_IV moulting. In the future, more research should work to uncover the molecular mechanism of J_IV moulting into adults by exploring the 19 differential genes.

DA is the final signal chemical, the ligands for DAF-12, and promotes reproduction and inhibits dauer programs. The dauer recovery of C. elegans is related to several genes, including GPCRs, daf-11, daf-2, daf-16, daf-7, daf-1/4, daf-8, daf-14, daf-9, and daf-12, belonging to the TGF-β/insulin signal pathway [13]. However, in B. xylophilus, all of these genes were not significantly up- or down-regulated in the transcriptome analysis of J_IV promoted by β-pinene. DA or ketoconazole had no function on the transformation of J_IV into an adult either (Figure 1). These results indicated that J_IV moulting into an adult might not be regulated by the TGF-β/insulin signal pathway. Another new signal pathway related to the 19 differential genes analysed by the transcriptome might be the reason for J_IV moulting. PKc-like superfamily genes might be responsible for the J_IV moulting of B. xylophilus. There is another possibility that the TGF-β/insulin signal pathway has not been regulated in J_IV at 6 h of contact with 1% β-pinene, which indicates that more development time is needed.

In conclusion, 19 genes—including 4 sterol hydroxylase, 5 dehydrogenase, 2 glucuronosyltransferase, 5 nuclear-related factor, 1 calcium-binding protein, 1 nitrogen metabolic regulation protein, and 1 cystathionine gamma-lyase—were related to J_IV moulting of B. xylophilus caused by β-pinene. Sterol hydroxylase, dehydrogenases, and glucuronosyltransferases might be responsible for the energy supply and histogenesis of J_IV moulting. However, the signal pathway of this moulting is not clear based on the rare significant differential signal genes, such as PKc-like superfamily genes. For pest control by preventing dauer moulting of B. xylophilus into adults who harm healthy pines, the key genes associated with moulting should be identified. Future research on the signal pathway of B. xylophilus’s dauer moulting will focus on these signal genes.