Pest Management Science - 2019 - Lu - Transcriptomic Profiling of Effects of Emamectin Benzoate On The Pine Wood Nematode

15264998, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ps.5575 by Cochrane Portugal, Wiley Online Library on [22/11/2022].
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Research Article
Received: 6 June 2019 Revised: 25 July 2019 Accepted article published: 6 August 2019 Published online in Wiley Online Library: 31 August 2019
(wileyonlinelibrary.com) DOI 10.1002/ps.5575
Transcriptomic profiling of effects of

emamectin benzoate on the pine wood
nematode Bursaphelenchus xylophilus
Feng Lu,# Kai Guo,# Anliang Chen,# Shani Chen, Haiping Lin and
Xiang Zhou*
Abstract
BACKGROUND: Emamectin benzoate (EB) has recently been successfully applied as a trunk injection for preventative control
of the pine wilt disease (PWD) caused by Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle. Here, a whole-organism
transcriptomic analysis provides comprehensive insights into the adverse effects of EB on B. xylophilus.
RESULTS: A large set of differentially expressed genes (DEGs) were found, demonstrating the antagonistic effects of EB on B.
xylophilus embryonic and larval development, reproduction, nervous and motor systems, and pathogenesis. In toxicity assays
with EB, the number of eggs laid, hatching rate, thrashing frequency, and developmental rate of B. xylophilus were significantly
suppressed at low concentrations (0.1 𝛍g mL−1 ). Moreover, the transcriptional changes validated by real-time quantitative PCR
showed downregulated transcript levels of the genes encoding pectate lyases, 𝜷-1,4-endoglucanases, and upregulated the
genes encoding glutamate-gated chloride channel, 𝜸-aminobutyric acid type 𝜷 receptor, uridine 5′ -diphospho-glucuronosyl
transferase, ATP-binding cassette transporter. The potential responses of B. xylophilus to EB included the upregulation of several
genes putatively contributing to oocyte protection, stem cell renewal, and xenobiotic degradation, implying the potential for
drug resistance to develop.
CONCLUSION: Our findings further our understanding of the effects of EB for managing the PWD and may help to improve the
pesticide-use strategies for controlling B. xylophilus.
© 2019 Society of Chemical Industry
Supporting information may be found in the online version of this article.
Keywords: plant-parasitic nematode; mode-of-action; nematicide; parasitism; trunk injection
1 INTRODUCTION There are few effective control measures available for PWD. Con-
The pine wood nematode (PWN) Bursaphelenchus xylophilus trol measures that have received attention include breeding resis-
(Steiner & Buhrer) Nickle can cause severe withering of Pinus trees, tant pine trees, microbial and chemical control of vector bee-
and induce outbreaks of pine wilt disease (PWD) in pine forests.1 tles, removing PWD-killed trees, fumigation of wood-packaging
PWN is an invasive species that originated in North America but materials, and trunk implantation.13,14 Tree-trunk injection is an
has spread to East Asia and Europe.2,3 Dispersal of the nematode environmentally-friendly method to precisely deliver chemical
can be natural via flying vectors such as cerambycid beetles agents for pest control. Tree-trunk injection with the compounds
(Monochamus), and artificial via wood-packaging materials used emamectin benzoate (EB), mesulfenfos, abamectin, or acetamiprid
has been practiced in southern China, Japan, and Portugal for
in domestic and overseas freight transport.4,5 In China, PWD has
many years to prevent PWN infestation.15,16 In particular, EB has
killed billions of pine trees since its introduction in 1982 resulting in
approximately 5 million m3 of annual wood loss.6 PWN reproduces
rapidly in host plants, developing to the adult stage through four
juvenile stages (J1–J4; nota bene: J1 molts to J2 inside egg) within ∗ Correspondence to: X Zhou, Collaborative Innovation Center of Zhejiang
less than a week.7 It has high fecundity with 90–100 eggs laid Green Pesticide, State key Laboratory of Subtropical Silviculture, School of
per female and can be highly concentrated with millions of PWN Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, Peo-
in host plants.7 PWN can secret numerous pathogenesis-related ple’s Republic of China. E-mail: xzhou@zafu.edu.cn
enzymes including cell-wall-degrading cellulases and pectate
# F Lu, K Guo, and A Chen joint first authors.
lyases, facilitating the penetration and migration through the
host cells.8–11 PWN can also induce dysfunction in the xylem and Collaborative Innovation Center of Zhejiang Green Pesticide, State key Labora-
tracheid, block the exchange of nutrients and water transport, tory of Subtropical Silviculture, School of Forestry and Biotechnology, Zhejiang
747
and ultimately causing tree death.12 A&F University, Hangzhou, People’s Republic of China
Pest Manag Sci 2020; 76: 747–757 www.soci.org © 2019 Society of Chemical Industry
15264998, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ps.5575 by Cochrane Portugal, Wiley Online Library on [22/11/2022]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
www.soci.org F Lu et al.
been found to be the most useful compound for trunk injec- transcripts influenced by the solubilizer and emulsifier. A water
tion against PWN, with 95% inhibitory rate (IC95 ) at a relatively control was also included with PWN adults exposed to (DW treat-
low concentration of 0.05 mg L−1 .15 The residual quantity of EB in ment). Each treatment contained three biological replicates for
most parts of pine trees was maintained at 0.04–1.91 μg g−1 for total RNA extraction.
27 months after tree-trunk injection, which is above the effective To extract total RNA from the 15 samples, the TRIzol Max Bac-
concentration of 0.031 μg g−1 .17 In China, ≤0.05% of Pinus massoni- terial RNA Isolation Kit (Thermo Fisher Scientific, New York, USA)
ana trees were killed by PWD 3 years after tree-trunk injection with was used following the manufacturer’s protocol. The RNA concen-
2% (20 g L−1 ) EB emulsifier agent.18 These results demonstrate that tration and purity were measured on a NanoDrop2000 (Thermo
EB is effective for the long-term control of PWD in forests. Fisher Scientific) and the integrity was checked by 1% agarose
The emamectin is a semi-synthetic derivative of abamectin (aver- gel electrophoresis and on the Agilent 2100 BioAnalyzer (Agilent,
mectin B1a and B1b ), a 16-membered macrocyclic lactone, pro- Santa Clara, CA, USA). The extracted RNA samples (approximately
duced by the fermentation of the actinomycete Streptomyces aver- 3 μg of total RNA per sample) were stored at −80 ∘ C and then sent
mitilis, and prepared as a salt with benzoic acid.19 The formulation to Woosen Co. (Hangzhou, China) for sequencing.22
of EB agents requires suitable adjuvants for solubilization because
EB has low water solubility. EB is a 𝛾-aminobutyric acid recep- 2.3 Assembly of transcriptome
tor (GABA-R) agonist and has insecticidal activity through inhibit- Nematode mRNA was enriched by Oligo (dT) magnetic beads
ing neurotransmission in arthropod pests.20 The glutamate-gated from each total RNA sample. Paired-end RNA-seq libraries of dif-
chloride channels in neuromuscular cells can be activated by EB, ferent treatments were prepared following the Illumina’s library
resulting in paralysis and death of insects and parasites. Similarly, construction protocol, and the libraries were then sequenced on
abamectin actively targets GABA-R; however, its IC95 against PWN an Illumina HiSeq 2500 platform (Illumina, San Diego, CA, USA).
is 0.16 mg L−1 , three-fold higher than that of EB.15 This implies that FASTQ files were produced and sorted. To obtain high-quality
EB induces other adverse effects on PWN viability. The modes of clean reads, raw reads were removed if they contained adapters,
action of EB in several arthropod and aquatic species have been had ≥10% of unknown nucleotides, and other low-quality indi-
investigated, such as EB interfering with molting and inducing cators. The remaining clean reads were mapped to a reference
apoptosis in the crustacean water flea Daphnia magna.21 However, genome (PRJEA64437, www.wormbase.org) using TopHat2 (ver-
its mode-of-action on PWN remains understudied, hindering fur- sion 2.0.3.12).
ther application in PWD control programs.
The objective of the present study was to profile the adverse
2.4 Identification of DEGs and gene annotation
effects of an EB trunk-injection agent on PWN by analyzing dif-
ferential transcripts from the nematode’s whole genome through The R package RSEM was used to calculate the fragments per
next-generation high-throughput sequencing. Our results provide kilobase of exon per million fragments mapped (FPKM) value.23
a detailed gene resource related to the control mechanism of EB DEGs between libraries were filtered out using the edgeR package
and may help in the development of control measures for PWD. (version 3.2.4) in BioConductor (release 2.12, R version 2.15.0).24
DEGs were identified with a fold change ≥2 and a false discov-
ery rate <0.001.25 The functions of DEGs were predicted by anno-
tation with several databases for further insight into the DEGs
2 MATERIALS AND METHODS involved in the toxicity of EB on PWN. The genes were annotated
2.1 Preparation of PWN and EB
by BLASTx search (E-value <10−5 ) against Wormbase, Swiss-Prot
The experimental PWN strain NB-6 was originally isolated from (www.uniprot.org), Gene Ontology (GO, www.geneontology.org),
diseased Pinus massoniana in the Ningbo area, Zhejiang province, Kyoto Encyclopedia of Genes and Genomes (KEGG; www.genome
China. PWN fed on 7-d-cultivated Botrytis cinerea cultured on .jp/kegg/kegg2.html) and Pathogen-Host Interaction (PHI) (ver-
potato dextrose agar (PDA) plates at 25 ∘ C. To synchronize PWN sion 4.2, www.phi-base.org) databases. Functional enrichment
adult stages, newly emerged second stage juveniles (J2) were analysis for GO terms and KEGG pathways was carried out using
collected and inoculated on B. cinerea plates in batches. After 3 d, the R package clusterProfiler.26
J2 developed into adults through juvenile stages (J3 and J4). The
Baermann funnel method was used to separate nematodes from
PDA plates, and PWN (10 000 nematodes mL−1 ) were collected by 2.5 Bioassays for assessment of the EB effects against PWN
centrifugation (4000 g) for 4 min. PWN adult fecundity, egg hatching rate, development rate, and
Technical grade EB (95% purity) was obtained from Zhejiang adult thrashing frequency were assessed among EB, ON, and
Shenghua Biok Biology Co. Ltd. (Huzhou, China). The applica- DW treatments. 1) Ten males and 10 females of PWN adults
ble formulation of 2% EB agent was prepared as 20 mg L−1 EB, were each transferred into 3 cm-diameter Petri dishes containing
30 mL L−1 n-butyl alcohol (solubilizer), 15 mL L−1 octylphenol 0.1 mg L−1 EB, ON, or DW, respectively. The number of eggs laid
polyoxyethylene-10 (emulsifier).18 Sterile distilled water (DW) was in each dish at 25 ∘ C was counted and recorded at 12-h intervals
used to dilute the agent to the desired EB concentrations. for 36 h and calculated as the number of eggs laid per female. 2)
The newly-laid eggs were collected 12 h after healthy nematodes
had mated. Then, 24-well tissue culture plates were used as culture
2.2 Total RNA extraction vessels, and approximately 50 eggs were added to each well
PWN adults (200 nematodes/sample) were collected after being and treated with 0.1 mg L−1 EB, DW, or ON at 25 ∘ C. Hatching
immersed in 0.1 mg L−1 EB for 12 h and 24 h at 25 ∘ C (EB12 and larvae were determined at 12-h intervals for 36 h and used to
EB24 treatments, respectively). PWN adults were also treated calculate the egg hatching rate. 3) PWN development rate was
with adjuvant concentration of octylphenol polyoxyethylene-10 determined by treating eggs with 0.1 mg L−1 EB, DW, or ON for
(15 mL L−1 ) and n-butyl alcohol (30 mL L−1 ) for 12 h and 24 h (ON12 24 h and then the eggs were transferred to PDA plates. The
748
and ON24 treatments, respectively) as a control for separating the proportions of adults were determined at 24-h intervals over
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Emamectin benzoate on B. xylophilus www.soci.org
Figure 1. A Venn diagram presenting the numbers of common and distinct differentially expressed genes (DEGs) among different RNA-seq libraries.
Swiss-Prot (UniProtKB/Swiss-Prot) symbol, GO (gene ontology) annotation or KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway of representa-
tive special DEGs with top fragment per kilobase per million fragments (FPKM) values in each area were presented. The up and down arrows marked on
the left of each DEG represent upregulation or downregulation, respectively.
4 days of incubation at 25 ∘ C. 4) A thrash of a nematode is 2.7 Data analysis

defined as a movement around the midpoint of the body, and The relative expression levels of selected DEGs were differentiated
thrashing frequency is an important indicator of PWN locomotion using a one-factor analysis of variance (ANOVA), and the number
and viability. PWN adults were treated with 0.1 mg L−1 EB, DW, of eggs laid, egg hatching proportions, adult proportions, and
or ON in 9-cm-diameter Petri dishes (100 adults/dish). In each thrashing frequency treatments with DW, ON, or 0.1 mg L−1 EB for
dish, 10 healthy adults were randomly selected for observation, 12–36 h were differentiated using a two-factor ANOVA. All analy-
and the number of thrashes per minute at 12-h intervals for 36 h ses were conducted using an updated version of DPS software.27
was recorded. All the above-mentioned bioassays included five
replicates.
3 RESULTS
2.6 qPCR analysis of selected DEGs 3.1 General features of PWN transcriptome assembly
and annotation
The RNA samples were extracted from PWN adults exposed
to DW, ON, and three concentrations of EB (0.05 mg L−1 , Following quality assessment and data filtering, the resultant tran-
0.1 mg L−1 , or 0.5 mg L−1 ) for 24 h. Real-time quantitative PCR scriptome of the 15 samples contained 959 741 626 clean reads
(qPCR) was performed to assess transcript levels of selected (Table S2). In total, 18 037 genes were predicted by mapping to
genes encoding pectate lyase (Wormbase ID: BXY_0827800), the PWN reference genome. After aligning the sequence similarity
𝛽-1,4-endoglucanase (BXY_0937800), glutamate-gated chlo- with several public databases, 12 272 (68.0%) genes were anno-
ride channel (BXY_0592100), 𝛾-aminobutyric acid type 𝛽 tated in Wormbase and 9693 (53.7%) genes were annotated in
receptor (BXY_0386600), uridine 5′ -diphospho-glucuronosyl Swiss-Prot; 4315 (23.9%) genes were subjected to GO classification,
transferase (BXY_1154300), and ATP-binding cassette trans- 3332 (18.5%) genes were annotated to 169 pathways according to
porter (BXY_0203900). For qPCR analysis, total RNA (1 μg) was KEGG, and 2420 (13.4%) genes were related to plant pathogenesis
reverse-transcribed into cDNA with a PrimeScript™ RT reagent Kit based on the PHI database.
with gDNA Eraser (TaKaRa, Tokyo, Japan). The qPCR of the cDNA
samples was performed using a SYBR Green PCR (SYBR Premix Ex 3.2 Identification and classification of DEGs
Taq™ II; TaKaRa). Paired primers were designed and are listed in There were only seven DEGs found between RNA-seq libraries
Table S1. PCRs were performed on a real-time PCR thermal cycler of DW, ON12, and EB12. These DEGs had functions in xenobiotic
(qTOWER 2.2, Analytik Jena, Analytikjena, Germany) under the biodegradation and developmental processes (Table S3). The EB
following conditions: 95 ∘ C for 30 s, followed by 40 cycles at 95 and ON treatments exerted significantly increasing effects on PWN
∘ C for 5 s and 60 ∘ C for 30 s. Data were analyzed using qPCRsoft gene expression in the next 12 h. Between the libraries at different
1.1 (Analytik Jena). Transcript quantification for each gene was time points for EB and ON treatments, or at the same time point of
performed using at least three independent replicates. Relative 24 h, a total of 5741 DEGs were filtered out.
fold-change in gene expression was normalized to the elongation Between EB24 and ON24 libraries, 758 upregulated and 562
749
factor 1-alpha (ef-1𝛼) gene (BXY_0569100). downregulated genes were found. The 241 GO-annotated DEGs
Pest Manag Sci 2020; 76: 747–757 © 2019 Society of Chemical Industry wileyonlinelibrary.com/journal/ps
Table 1. The Bursaphelenchus xylophilus life cycle-related DEGs with top FPKM values in EB24 vs. ON24a
FPKM
Wormbase ID Annotation EB24 ON24 Log2 (FC)b GO/KO ID
Larval development and programmed cell death

BXY_0684700 NHL repeat protein 243.25 89.52 1.44 GO:0009791
BXY_0640900 Glutamine amidotransferase and Sugar isomerase domain 345.13 132.21 1.38 GO:0002164
BXY_0740900 ATP-dependent RNA helicase cgh-1 670.04 280.90 1.25 GO:0002164
BXY_1544800 Homeobox protein vab-15 4.23 1.84 1.18 GO:0019953
BXY_1442600 Transketolase, thiamine diphosphate binding domain protein 150.72 310.43 −1.04 GO:0002164
BXY_1659300 Inactive angiotensin-converting enzyme 6.95 16.17 −1.22 GO:0042303
BXY_1542000 Protein disulfide isomerase 171.28 425.76 −1.31 GO:0002164
BXY_1181600 Protein Lon-1 8.87 26.27 −1.56 GO:0048589
Cuticle collagen
BXY_1087500 Collagen protein 68 442.09 165.78 1.42 /
BXY_0975000 Cuticle collagen 40 95.37 37.37 1.35 GO:0048856
BXY_1769800 Cuticle collagen 6 263.24 550.24 −1.06 /
BXY_0412000 Cuticle collagen 19 97.92 217.47 −1.15 /
BXY_0590000 Collagen 2 108.94 254.09 −1.22 /
BXY_0075100 Collagen 5 36.74 86.56 −1.24 GO:0005198
BXY_1699500 Nematode cuticle collagen domain protein 48.84 121.57 −1.32 /
BXY_0651700 Collagen alpha-5(IV) chain 69.45 176.89 −1.35 GO:0048856
BXY_1203100 Nematode cuticle collagen domain protein 23.47 77.86 −1.73 GO:0048856
Reproduction
BXY_0953700 G2/mitotic-specific cyclin-B1 218.30 87.58 1.32 KO04914
BXY_0997200 KH domain protein 388.85 160.00 1.28 GO:0048569
BXY_0117200 Pumilio-family RNA binding repeat containing protein 222.77 92.03 1.28 GO:0019953
BXY_1002100 Ribosomal protein S6 kinase 𝛼-1 90.04 39.84 1.18 KO04212
BXY_1561800 Major sperm protein 464.16 210.88 1.14 GO:0044237
BXY_1730000 Sperm-specific class P protein 19 19.50 8.86 1.14 GO:0046983
BXY_0723800 Major sperm protein domain 64.00 30.89 1.05 /
BXY_0363400 Papilin 74.83 160.21 −1.10 GO:0000003
BXY_0402700 Pregnancy associated glycoprotein 3 545.00 1249.67 −1.20 KO04974
BXY_0603100 50 kDa hatching enzyme 0.70 1.70 −1.28 GO:0003824
BXY_0100900 Membrane-associated tyrosin- and threonine-specific cdc2-inhibitory kinase 2.87 8.39 −1.55 GO:0007276
BXY_0255000 Estradiol 17-𝛽-dehydrogenase 12 5.03 17.22 −1.78 GO:0003006
BXY_0364600 Hydroxyacyl-coenzyme A dehydrogenase 112.72 781.96 −2.79 GO:0050796
Aging
BXY_1563600 Heat shock protein 70 b2 120.00 26.71 2.17 GO:0007568
BXY_0640100 CRE-HSP-70 protein 238.58 61.91 1.95 GO:0007568
BXY_0811600 Bifunctional 3′ -phosphoadenosine 5′ -phosphosulfate synthase 266.08 122.12 1.12 GO:0007568
BXY_1442600 Transketolase, thiamine diphosphate binding domain protein, partial 150.72 310.43 −1.04 GO:0007568
BXY_1542000 Peptide transporter family 1 8.04 22.69 −1.50 GO:0002164
a EB24 refers to the library constructed by transcripts of B. xylophilus treated by EB agent for 24 h. ON24 refers to the library constructed from adjuvant
treatment for 24 h. Transcript level is expressed in fragments per kilobase per million fragments (FPKM) values.
b FC means fold change of differentially expressed genes (DEGs) between the two libraries.
were classified into 41 classes (level 2 subcategories) in the three KEGG-annotated DEGs were classified into 18 pathways (Table S4),
ontologies of molecular function (15 classes), cellular component mainly including lysosome, drug metabolism, and metabolism of
(nine classes) and biological process (17 classes). The largest class xenobiotics by cytochrome P450, indicating the PWN response to
of DEGs was single-organism process (80 DEGs upregulated and 82 EB toxicity. Pairwise comparisons of ON24 vs. ON12 and EB24 vs.
downregulated) in the biological process ontology (Fig. S1(A)). All EB12 showed different numbers of DEGs subjected to GO terms
DEGs in the classes: molecular function regulator, molecular trans- and pathways (Figs. S1 and S2), demonstrating the different effects
ducer activity, signal transducer activity, synapse part, synapse, cell from ingredients and exposure time.
junction, and antioxidant activity were upregulated. Those in the There were different numbers of common and distinct DEGs
classes: extracellular matrix, extracellular region part, extracellu- among different treatments (Fig. 1; nota bene: the symbols and
lar region, and extracellular matrix component were downregu- abbreviations are listed in Table S5). For example, 312 (5.46%) DEGs
lated. This demonstrated that EB treatment particularly affected between EB24 and ON24 libraries were distinct, 1416 (24.78%)
750
the PWN neurotransmission and extracellular matrix. The 185 DEGs between EB12 and EB24 libraries were distinct, and 1602
Table 2. The Bursaphelenchus xylophilus nervous and motor system-related DEGs with top FPKM values in EB24 vs. ON24a
FPKM
Wormbase ID Annotation EB24 ON24 Log2 (FC)b GO/KO IDa
Neuron and muscle structure

BXY_1598600 Adenosine receptor homolog 9.87 4.02 1.30 KO04080
BXY_1644800 BMA-TIR-1, isoform e 28.02 11.61 1.27 GO:0005488
BXY_1596600 Myosin and Myosin head and Myosin tail domain containing protein 175.27 73.18 1.26 GO:0030182
BXY_0593200 Serpentine receptor class r-10 3.61 1.62 1.16 GO:0016020
BXY_0143800 Acetylcholine receptor subunit 𝛽-type acr-2 14.11 6.64 1.09 GO:0007268
BXY_0909500 Unc-49B protein 8.11 3.99 1.02 KO04080
BXY_1320900 Variant SH3 domain UNC89 5.38 2.60 1.05 GO:0055001
BXY_0522200 Glycogen muscle form 30.01 94.09 −1.65 GO:0005488
BXY_0358300 Asparagine synthetase 6.55 16.17 −1.22 GO:0051716
BXY_1121200 Cell death specification protein 2 3.89 8.92 −1.19 GO:0008219
Ion channel
BXY_0592100 Glutamate-gated chloride channel 2.14 0.92 1.22 GO:0006811
BXY_0738200 Ligand-gated ion channel 50 12.43 5.56 1.16 GO:0006811
BXY_0354000 Ligand-gated ion channel 50 13.41 6.20 1.11 GO:0006811
BXY_1626300 Voltage-dependent calcium channel gamma-3 subunit 12.47 5.99 1.08 GO:0006811
BXY_0865900 Amiloride-sensitive sodium channel subunit beta 8.22 16.65 −1.02 GO:0006811
Response to stimulus, chemical or stress
BXY_1563600 Heat shock protein 70 b2 120.00 26.71 2.17 GO:0050896
BXY_0768000 CRE-HSP-70 protein 207.57 60.14 1.79 GO:0050896
BXY_1186000 Homeobox domain CEH14 25.38 12.14 1.06 GO:0009266
BXY_1439800 Dimethylaniline monooxygenase 30.53 68.42 −1.16 GO:0006950
BXY_1529500 EF hand domain containing protein 13.36 31.05 −1.22 KO04114
BXY_0853200 CRE-RHY-1 33.79 15.78 1.10 GO:0006950
BXY_1542000 Protein disulfide isomerase 171.28 425.76 −1.31 GO:0050896
BXY_0673600 Flavin-containing monooxygenase FMO domain containing protein 172.64 453.73 −1.39 GO:0050896
BXY_0522300 Lysozyme 13.58 63.02 −2.21 GO:0006950
BXY_0364600 Hydroxyacyl-coenzyme A dehydrogenase 112.72 781.96 −2.79 GO:0042221
(28.04%) DEGs between ON12 and ON24 libraries were distinct. response to external stimulus(GO:0009605; 6 DEGs), response to
The DEGs with top FPKM values between EB24 and ON24 libraries chemical (GO:0042221; 7), and response to stress (GO:0006950; 17)
included those encoding major sperm proteins (Swiss-Prot sym- (Tables 2 and S6).
bol: MSP78), hydrolase (HYEP), cuticle collagen 40 (COL13), infertile There were 20 DEGs subjected to KO terms (KO00980:
crescent (DEGS1), and HSP-70 protein (HSP74). metabolism of xenobiotics by cytochrome P450 and KO00982:
drug metabolism, cytochrome P450) encoding different hydro-
lases, glutathione S-transferase and UPD-glucuronosyltransferase
3.3 DEGs related to physiological and pathogenic
(Tables 3 and S6), likely related to the detoxification process.
characteristics of EB-treated PWN
Based on the PHI database, the DEGs related to pathogenesis
EB exerted effects on transcripts related to embryonic develop-
encoding different pectate lyases were downregulated (Table 4),
ment (GO:0009790; 39 DEGs), larval development (GO:0002119; 5),
and all six DEGs encoding 𝛽-1,4-endoglucanases related to cel-
molting cycle (GO:0042303; 13), aging (GO:0007568; 19), sex dif- lulose degradation were also downregulated. Other potential
ferentiation (GO:0007548; 5), and reproduction (GO:0000003; 30). pathogenesis-related genes, such as those encoding venom
Except for eight out of the 11 DEGs subjected to programmed allergen and expansin, many of which were downregulated,
cell death (GO:0012501) that were upregulated, many of the implied an attenuation effect of EB on PWN pathogenicity.
development-related DEGs were downregulated in the EB treat-
ment. Of the 29 DEGs encoding nematode cuticle collagen 27 were
downregulated (Tables 1 and S6). 3.4 Bioassays for assessment of the EB effects against PWN
In EB treatment, many of DEGs related to the nervous system The EB treatment significantly reduced the number of PWN eggs
were upregulated, including neuron-neuron synaptic transmission laid (F 2,36 = 55.39, P < 0.001), egg-hatching rates (F 2,36 = 46.48,
(GO:0007270; 1 DEG), nervous system development (GO:0007399; P < 0.001), adult proportion (F 2,36 = 175.75, P < 0.001), and thrash-
6), chloride transport (GO:0006821; 3), and neuron differentiation ing frequency (F 2,36 = 8.03, P = 0.0013) compared with those of
751
(GO:0030182; 5). Meanwhile, EB affected transcripts related to the DW and ON treatments (Fig. 2(a)–(d)). These inhibitory effects
Table 3. The Bursaphelenchus xylophilus xenobiotics biodegradation-related DEGs with top FPKM values in EB24 vs. ON24a
FPKM
Wormbase ID Annotation EB24 ON24 Log2 (FC)b GO/KO IDa
Hydrolase
BXY_0557700 Hydrolase, 𝛼/𝛽 domain protein 5.95 12.84 −1.11 KO00980
BXY_1009100 Hydrolase, 𝛼/𝛽 domain protein, partial 14.78 32.11 −1.12 KO00100
BXY_0780900 Amidohydrolase 1 domain containing protein 40.53 90.65 −1.16 KO00983
UDP-glucosyl transferase
BXY_1154300 UDP-glucuronosyl and UDP-glucosyl transferase 96.92 41.87 1.21 KO00980
BXY_0209700 UDP-glucosyl transferase domain 62.63 29.58 1.08 KO00980
BXY_1641400 UDP-glucuronosyl/UDP-glucosyltransferase family 34.65 70.64 −1.03 KO00980
BXY_1088400 UDP-glucuronosyltransferase 2C1 5.54 11.48 −1.05 KO00980
BXY_1088300 UDP-glucuronosyltransferase 2C1 1.87 4.36 −1.22 KO00980
Gut esterase
BXY_0546000 Gut esterase 1 4.83 2.03 1.25 KO01044
Dehydrogenase
BXY_0016000 Trans-1,2-dihydrobenzene-1,2-diol dehydrogenase-like isoform X3 47.65 108.46 −1.19 KO00980
Glutathione S-transferase
BXY_1477400 Glutathione S-transferase 1 8.43 2.57 1.71 KO00980
BXY_0629100 Sigma class glutathione S-transferase 20.97 9.90 1.08 KO00980
BXY_1154500 Glutathione S-Transferase GST4 16.56 7.99 1.05 /
BXY_0299600 Glutathione S-transferase 1 284.75 681.05 −1.26 KO00980
BXY_0299100 Glutathione S-transferase, GST6 2.47 15.38 −2.64 KO00980
ABC transporter
BXY_0203900 Haf ABC transporter 4 13.82 4.58 1.59 GO:0022857
Peroxidase
BXY_1758800 Peroxidase skpo-1 29.79 14.25 1.06 /
BXY_1747900 Peroxiredoxin-1 11.76 5.8 1.02 GO:0042743
increased with EB-treatment time: egg laying (F 2,36 = 145.25, homologs studied in the model nematode Caenorhabditis ele-
P < 0.001), hatching rate (F 2,36 = 443.94, P < 0.001), and thrashing gans. In bioassays, the number of eggs laid, hatching rate and
frequency (F 2,36 = 334.25, P < 0.001). There were no significant developmental rate significantly decreased under EB treatment,
differences between DW and ON treatments, demonstrating the consistent with a set of DEGs involved in germline proliferation,
adjuvants did not have nematicidal activity. larval development, and reproduction. A gene homolog of vab-15
involved in embryonic lethality and defective egg laying in C. ele-
3.5 Validation of RNA-seq expression data by qPCR gans was upregulated following EB treatment, possibly related to
The relative expression levels of the selected DEGs in PWN treated the reduced number of eggs laid and hatching rate.28 In C. ele-
with different concentrations of EB were evaluated by qPCR (Fig. 3). gans, an initial step of embryonic development involves the sperm
The expression levels of all selected DEGs differed significantly stimulating V-ATPase activity in oocytes by signaling the degrada-
(pectate lyases: F 2,10 = 151.9, P < 0.001; 𝛽-1,4-endoglucanse: tion of GLD1 (KH domain protein, a translational repressor blocking
F 2,10 = 735.2, P < 0.001; glutamate-gated chloride channel: V-ATPase synthesis).29 Thus, the upregulated gene encoding GLD1
F 2,10 = 35.0, P < 0.001; 𝛾-aminobutyric acid type 𝛽 receptor: may contribute to the decreased hatching rate and growth rate of
F 2,10 = 15.8, P < 0.001; uridine 5′ -diphospho-glucuronosyl trans- larvae hatching from EB-treated eggs. Another EB-induced gene
ferase: F 2,10 = 17.0, P < 0.001; ATP-binding cassette transporter:
encodes NHL repeat proteins, which is homologous with lin41 in C.
F 2,10 = 23.1, P < 0.001). These results are consistent with those
elegans and is also a translational repressor, playing a vital role in
shown in the corresponding transcriptome data.
regulating the developmental timing of seam cells and epidermal
blast cells.30
4 DISCUSSION More genes potentially involved in nematode development
In the present study, a detailed transcriptome analysis and toxic- were downregulated. The functioning proteins included: a papilin
ity assays were performed, demonstrating that low-dose EB treat- PPN1 which plays a role in embryogenesis31 ; ACN1 which acts
ment exerted adverse effects on PWN development, reproduction, downstream of the heterochronic protein LIN-41 to regulate
and motion. postembryonic cell division; PDI-2 which is a disulfide isomerase
There is a lack of previous studies on PWN gene functions; involved in cuticle synthesis and molting32 ; LON-1 which is
752
therefore, the present study depends on predictions based on expressed in hypodermal tissue to regulate C. elegans body size
Table 4. The Bursaphelenchus xylophilus pathogenesis-related DEGs values in EB24 vs. ON24a
FPKMb
Wormbase ID Annotation EB24 EC24 Log2 (FC)b PHI IDa
BXY_1090600 Pectate lyase 39.22 106.65 −1.44 /

BXY_1260300 Pectate lyase 14.93 106.61 −2.84 PHI:4620
BXY_1378100 Venom allergen-like protein VAP2 1.30 0.36 1.87 /
BXY_1026500 Venom allergen-like protein 1 2.47 1.05 1.23 /
BXY_0791800 Venom allergen-like protein 5.40 11.12 −1.04 /
BXY_0809000 Venom allergen-like protein VAP3 6.27 14.94 −1.25 /
BXY_0693500 𝛽-1,4-endoglucanase 1.44 3.06 −1.09 /
BXY_1576900 Expansin like protein 1.01 0.47 1.11 /
BXY_1576500 Expansin like protein 9.48 3.03 1.64 /
BXY_1576600 Expansin like protein 0.15 2.42 −3.98 /
treatment for 24 h.
b Transcript level is expressed in fragments per kilobase per million fragments (FPKM) values. FC means fold change of differentially expressed genes
(DEGs) between the two libraries.
determination33 ; hydroxyacyl-coenzyme (HCDH) which is a dehy- protein (SARM1) is important for neuronal differentiation in
drogenase involved in insulin secretion regulation; a transketolase C. elegans.39 TIR-1 acts downstream of a voltage-gated cal-
TKTL2 which is predicted to function in cell proliferation34 ; LET767 cium channel and assembles a synaptic signaling complex that
which is a very-long-chain 3-oxooacyl-coA reductase that was regulates odorant receptor expression. Its overexpression might
downregulated causing defects in embryogenesis, female repro- cause PWN nervous system disorder in response to chemical
duction, and molting behavior in C. elegans; and PMY13 which is a stimuli.
cdc2-inhibitory kinase regulating oocyte maturation.35 Few nervous system-related genes were found to be downreg-
Our results indicate that EB exerted different effects on the ulated. The gene frh-1 encoding the EF hand domain protein was
nematode nervous and motor system, and that most genes were downregulated causing reduced lifespan and sensitivity to oxida-
upregulated. Besides glc-1 (gene encoding glutamate-gated tive stress in C. elegans and might have similar effects on PWN.40
chloride channel), DEGs related to ion channels were overex- Glutamine-rich domains are hallmarks of some neurodegenerative
pressed, similar to that reported in the EB toxic mechanism in disease proteins.41,42 The gene encoding ASNS (asparagine syn-
Daphnia magna.21 In nematodes, the muscle arm termini harbor thetase and glutamine hydrolyzing) were downregulated, which
the post-synaptic elements of the neuromuscular junction.36 might cause glutamine richness in PWN cells to aggravate the ner-
The upregulated gene encoding myosin-4, which interacts with vous system.
MADD-4 guiding muscle arm extension in C. elegans, may cause Multiple environmental sensor-related genes were influ-
disorder of the muscular contractions resulting in lower thrashing enced. The gene ceh-14 is necessary for C. elegans thermo-
frequency, which would lead to PWN paralysis. UNC-89 (mus- tactic behavior.43 Upregulated ceh-14 might cause PWN to be
cle M-line assembly protein) interacts with paramyosin, and more sensitive to elevated temperatures. RHY-1 (regulator of
the overexpressed SH3 domain of UNC-89 caused paramyosin hypoxia-inducible factor 1) inhibits HIF-1, and HIF-1 functions
mislocalization,37 and thus the upregulated unc-89 might cause as an O2 sensor for adaptation to hypoxia through the EGL-9
PWN disorder of muscle architecture. The upregulated acr-2 gene pathway. Thus, the upregulated rhy-1 in PWN might cause dis-
might cause disorder in the functioning of cholinergic or GABAer- order in response to hypoxia stress in plants.44 All 13 serpentine
gic motor neurons, which encode acetylcholine receptor subunit receptor-coding genes were upregulated, which might influence
𝛽-type that is important for the coordinated excitation and inhibi- the diversity of the olfactory neurons and nematode chemotaxis.
tion of body muscles underlying sinusoidal movement.38 The gene PWN has a larger number of detoxification enzymes than
753
tir-1 encoding Toll-interleukin-1 receptor (TIR) motif-containing other plant-parasitic nematodes, including expanded families
Figure 2. Pine wood nematode (PWN) bioassays in the treatments of emamectin benzoate (EB), distilled water (DW) and adjuvant control (ON). (a)
Cumulative numbers of eggs laid per female. (b) The hatching rate of eggs. (c) The proportions of adults hatched from treated eggs. (d) The thrashing
frequency (times per min). Different lowercase letters marked on the bars indicate significant differences (Fisher’s LSD, P < 0.05). Error bars: SEM from five
replicates.
of lysosome pathway genes and cytochrome P450 pathway in C. elegans. The upregulated rskn-1 and fbf-2 might be involved
genes.10 In our study, many of the hydrolase-related genes in PWN recovery through stem cell renewal. The upregulated
were downregulated, such as those encoding amidohydro- gab-1 encoding 𝛾-aminobutyric acid receptor subunit 𝛽 (GABA,
lase, glycoside hydrolase, epoxide hydrolase, and esterase. an inhibitory neurotransmitter) were indicated to be involved in
UDP-glucuronosyltransferase and glutathione S-transferases ivermectin resistance and EB resistance in salmon lice and might
of the class GST1 (GST 𝜋) are important phase II detoxification be involved in potential EB resistance in PWN.48,49
enzymes. GST1s conjugate compounds to glutathione to reduce In the present study, many of the putative pathogenesis-related
cellular oxidative stress and are also involved in modulating the genes for PWN parasitism in host plants were downregulated,
vulnerability of dopamine signaling neurons in C. elegans.45 The implying negative effects of EB on PWN infectivity. All the
GST1-coding gene was downregulated in PWN, which might genes encoding pectate lyases (PHI: 4620) were downregu-
reduce the ability to encounter exogenous EB. lated, which are secreted into plant tissues to help feeding and
Embryo and oocyte protection, stem cell renewal, and drug migration in the tree.8 All genes involved in the endohydrolysis of
resistance-related genes were upregulated. For example, EGG6 1,4-𝛽-D-glucosidic linkages in cellulose were also downregulated.
encoding leucine-rich repeat proteins was upregulated, which The disruption of calcium signaling caused by Ca2+ influx might
contributes to the embryonic sheath and cuticular apical extra- decrease the expression of these genes.49 Hydrogen peroxide
cellular matrix. In C. elegans, CGH-1 (ATP-dependent RNA heli- accumulation promoted the pathogenicity of PWN.50 In the cur-
case) prevents the physiological germline apoptosis from killing rent results, another possibility is the induced peroxidase-coding
all developing oocytes.46 The upregulated gene encoding CGH-1 genes such as SKPO1 and PRDX (peroxiredoxin-1) that lower the
in our results might function in the PWN response to protect concentration of H2 O2 , and might be also related to the down-
oocytes from EB-induced overexpression of genes involved in pro- regulation. Moreover, venom allergen-like proteins (VAPs) are a
grammed cell death. The gene rskn-1 encodes ribosomal protein type of cysteine-rich secretory protein, involved in the migration
S6 kinase 𝛼-1 (KS6A1), which regulates spermatocyte dedifferenti- of PWN inside the host by suppressing the pine tree defences.51,52
ation in C. elegans.47 The gene fbf-2 encodes fem-3 mRNA-binding Two of six VAP-coding genes were downregulated under EB treat-
754
factor 2 and is involved in the self-renewal of germline stem cells ment. One of three genes encoding expansin proteins involved in
Figure 3. Relative expression levels of the selected differentially expressed genes (DEGs) in pine wood nematode (PWN). Five cDNA samples were derived
from PWN treated with distilled water (DW), adjuvant mixture (ON, control), 0.05, 0.1, 0.5 mg L−1 emamectin benzoate (EB). (a) DEG encoding pectate
lyase (BXY_0827800). (b) DEG encoding 𝛽-1,4-endoglucanase (BXY_0937800). (c) DEG encoding glutamate-gated chloride channel (BXY_0592100). (d)
DEG encoding 𝛾-aminobutyric acid type 𝛽 receptor (BXY_0386600). (e) DEG encoding uridine 5′ -diphospho-glucuronosyl transferase (BXY_1154300). (f )
DEG encoding ATP-binding cassette transporter (BXY_0203900). Different lowercase letters marked on the bars indicate significant differences (Fisher’s
LSD, P < 0.05). Error bars: SEM from three biological replicates.
loosening non-covalent interactions of components in plant cell the grants from the National Natural Science Foundation of China
walls to facilitate enzymatic activities were also downregulated.9 (Grant Nos.: 31870637 and 31200487), the 111 Project (D18008)
In conclusion, our results provide a genetic dataset for obtaining and Zhejiang Key Research Plan (2019C02024 and 2016C32016).
global insights into the adverse effects of EB on PWN. A set of
functional genes related to PWN viability and pathogenicity were
found downregulated, helping towards further development of SUPPORTING INFORMATION
PWD chemical control measures. Supporting information may be found in the online version of this
article.
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15264998, 2020, 2, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ps.5575 by Cochrane Portugal, Wiley Online Library on [22/11/2022].

(wileyonlinelibrary.com) DOI 10.1002/ps.5575

Transcriptomic proﬁling of eﬀects of

Supporting information may be found in the online version of this article.

Keywords: plant-parasitic nematode; mode-of-action; nematicide; parasitism; trunk injection

4 days of incubation at 25 ∘ C. 4) A thrash of a nematode is 2.7 Data analysis

Wormbase ID Annotation EB24 ON24 Log2 (FC)b GO/KO ID

Larval development and programmed cell death

Wormbase ID Annotation EB24 ON24 Log2 (FC)b GO/KO IDa

Neuron and muscle structure

BXY_1090600 Pectate lyase 39.22 106.65 −1.44 /

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