The pine sawyer beetle, Monochamus alternatus Hope, is a devastating wood borer of several species of pine trees, and the main transmitting vector of the pine wood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle (Aphelenchida: Parasitaphelenchidae). To explore new techniques for prevention and control of this destructive beetle, a novel gene vacuolar ATPase subunit H (V-ATPase H) was chosen as RNA interference (RNAi) target gene. Relative expression of V-ATPase H in different tissues and silencing efficiency in an in vitro RNAi experiment was assayed by using reverse transcription–quantitative polymerase chain reaction. The results indicated that the mRNA abundance of V-ATPase H in the gut was significantly higher than that in fat body, residual body, and hemolymph. Double-stranded RNA (dsRNA) targeting V-ATPase H was able to silence the expression of target gene effectively at 24 h posttreatment. Expression of immunity-related genes was examined after treatment with dsRNA targeting V-ATPase H, and transcript levels were compared with the control. The results showed that RNAi suppression of V-ATPase H inhibited the expression of immunity-related genes. This is the first demonstration of an in vitro RNAi experiment in any insect hemolymph that provides a novel environment for evaluating RNAi in insects, as well as shows potential for developing RNAi-mediated strategy for the control of M. alternatus.

The pine sawyer beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae), is a devastating wood borer of several species of pine trees (e.g., Pinus massoniana Lamb., P. thunbergii Parl., P. taiwanensis Hayata, and P. densiflora Sieb. et Zucc.) (Xiao 1992). Moreover, it transmits a plant endoparasite pine wood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle (Aphelenchida: Parasitaphelenchidae), to uninfected pine trees through feeding and oviposition behaviors, which causes a serious disease known as pine wilt disease in host trees (Linit 1988, Mamiya and Enda 1972). Pine wilt disease has incurred tremendous damage to forest ecosystems and huge economic losses locally in Asia and Europe (Mota et al. 1999, Cheng et al. 1983, Mamiya and Kiyohara 1972, Robertson et al. 2011, Yi et al. 1989).

Management of M. alternatus became a new impetus for control of the disease because the beetle is the main vector of B. xylophilus. Notwithstanding, current and widely used strategies (e.g., trapping, spraying insecticides) for reducing the population density of M. alternatus are inadequate to achieve the goal of eliminating pine wilt disease (Ye 2019). Thus, new methods must be developed for prevention and control of both M. alternatus and the disease.

RNA interference (RNAi) technology has been considered as a potential insect pest management strategy by suppression of key enzymes or proteins of pest insects (Gu and Knipple 2013, Kola et al. 2015, Mezzetti et al. 2020, Zhang et al. 2017). Candidate RNAi target genes were screened and their functions were further studied in a variety of crop insect pests in ongoing attempts to develop a new generation of species-specific insecticides (Castellanos et al. 2019, Whyard et al. 2009, Zhu et al. 2016). Nevertheless, RNAi-based control methods have not been extensively explored against forest insect pests (Rodrigues et al. 2017).

In the present study, we report a novel RNAi target gene, vacuolar ATPase subunit H (V-ATPase H), in the pine sawyer beetle. The study was two-tiered with one avenue focused on the relative expression of V-ATPase H in different tissues (e.g., hemolymph, gut, fat body, and residual body) of M. alternatus larvae, and the other avenue directed to the silencing efficiency of double-stranded RNA (dsRNA) targeting V-ATPase H in an in vitro RNAi experiment in the hemolymph of M. alternatus larvae. Our overall goal was to establish expression of an immunity-related gene after treatment with dsRNA targeting V-ATPase H to establish the prospect of developing RNAi-mediated strategy for the control of M. alternatus.

Insect and tissue collection.Monochamus alternatus larvae were collected from the xylem of the host Pinus massoniana trees in Chaohu City, Anhui province, China, in April 2020. Larvae were kept individually in 5-ml centrifuge tubes without a food source at 8°C and 50% relative humidity until dissected. Fifth-instar mature larvae were chosen and used in the experiments. Individual larvae were dissected with sterile forceps and scissors. Hemolymph, gut, fat body, and the residual body of larvae were collected. Each tissue from one individual was used as one biological replicate with three replications.

Target gene selection. The global transcriptome of M. alternatus larvae was sequenced on the HiSeq 4000 platform (Illumina, San Diego, CA) at the Beijing Genomics Institute (Shenzhen, China); the RNA-seq data were deposited to National Center for Biotechnology Information (NCBI) with accession number SRR3742169. The transcriptome was constructed separately. The target gene V-ATPase H was chosen from the coding sequence database of M. alternatus according to previous reports on silencing efficiency of other V-ATPase subunits in other crop insects (Baum et al. 2007, Upadhyay et al. 2011, Yao et al. 2013).

dsRNA synthesis. Total RNA was extracted from M. alternatus larvae using Total RNA Kit I (Omega, Norcross, GA) following the manufacturer's instructions. The integrity of RNA samples was checked by electrophoresis on a 1% agarose gel, and the concentration of RNA was determined using a Multiskan GO microplate spectrophotometer with a lDrop ultramicro detection plate (Thermo Fisher Scientific, Waltham, MA). Complementary DNA (cDNA) was prepared from the total RNA using EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen, Beijing, China) according to the manufacturer's protocol. Fragments of target gene V-ATPase H and enhanced green fluorescent protein (EGFP) were amplified with the above cDNA and PEGFP-C1 plasmid (Miaolingbio, Wuhan, Hubei, China), respectively, by using TransStart KD Plus PCR SuperMix (TransGen). Primers for amplification of V-ATPase H and EGFP were designed by Primer3web (version 4.1.0) using the conserved domain of corresponding genes respectively (Table 1). The polymerase chain reaction (PCR) experiments were conducted on a PCR instrument (Langji, Hangzhou, Zhejiang, China). The resulting PCR product was checked on a 1% agarose gel, and the gel containing a band was cut out and purified with SanPrep Column DNA Gel Extraction Kit (Sangon, Shanghai, China). After sequencing on a 3730XL DNA Analyzer (Applied Biosystems, Forster, CA) by Sangon Biotech (Shanghai) Co., Ltd. to ensure the sequences, the purified PCR product was used as a template to synthesize the dsRNA of target gene and EGFP with MEGAscript RNAi Kit (Thermo Fisher Scientific).

Table 1

Primers for polymerase chain reaction.

Primers for polymerase chain reaction.
Primers for polymerase chain reaction.

In vitro RNAi. Hemolymph was collected from fifth-instar larvae. A volume of 1 lL of synthesized dsRNA (1,000 ng/lL) based on V-ATPase H or EGFP (control) was mixed with 20 lL of hemolymph in a nuclease-free tube. The mixture was subsequently maintained at 28°C. After 24 h, 48 h, or 72 h, the hemolymph was collected for the next step RNA extraction. There were five replicates for each time interval.

Reverse transcription–quantitative PCR (RT-qPCR). The total RNA Kit I (Omega) was used to extract total RNA from the samples, and the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen) was used to prepare cDNA. All procedures were performed according to the manufacturer's instructions. Gene-specific primers for V-ATPase H, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and immunity-related genes (e.g., lysozyme, TGF-beta activated kinase 1 [TAK1], and pelle) amplification were designed on Primer3web (version 4.1.0) (Table 2). Triple cDNA samples were amplified on a LineGene 4800 Fluorescent Quantitative PCR Detection System (Bioer, Hangzhou, Zhejiang, China) following the protocol of PerfectStart Green qPCR SuperMix (TransGen). The PCR program was set as: 94°C for 30 s, followed by 40 cycles of 94°C for 5 s and 60°C for 30 s. The specificity of the SYBR green PCR signal was confirmed by a melting curve from 60°C to 94°C. The GAPDH was used as a reference gene for an internal RNA control. Relative mRNA accumulation was determined by the 2–ΔΔCT method (Livak and Schmittgen 2001).

Table 2

Primers for quantitative polymerase chain reaction.

Primers for quantitative polymerase chain reaction.
Primers for quantitative polymerase chain reaction.

Statistical analysis. The relative expression of V-ATPase H in different tissues was analyzed by one-way analysis of variance, followed by Tukey's test. The Student's paired-samples t test was used to compare transcript levels between treatment and control groups. All the data were analyzed by SPSS 19.0 software.

Relative expression of V-ATPase subunit H gene in different tissues. The relative expression of V-ATPase H in different tissues (e.g., hemolymph, gut, fat body, residual body) of M. alternatus larvae was observed with RT-qPCR technology. The results showed that there were significant differences on the relative expression of V-ATPase H among different tissue samples (F=34.151; df= 3, 8; P < 0.001). The relative expression of V-ATPase H in gut was significantly higher than that in fat body, residual body, and hemolymph (Fig. 1).

Fig. 1

Relative expression of vacuolar ATPase subunit H (V-ATPase H) in different tissues of M. alternatus larvae. (Error bars are SE. The different letters labeled above the bars represent significant differences between different tissues at the P = 0.05 level by ANOVA, while the same letters represent no difference.)

Fig. 1

Relative expression of vacuolar ATPase subunit H (V-ATPase H) in different tissues of M. alternatus larvae. (Error bars are SE. The different letters labeled above the bars represent significant differences between different tissues at the P = 0.05 level by ANOVA, while the same letters represent no difference.)

Close modal

Silencing efficiency in hemolymph.In vitro RNAi experiment was performed in M. alternatus larval hemolymph. The transcript level of V-ATPase H was tested at 24 h, 48 h, and 72 h posttreatment to study the silencing efficiency of dsRNA targeting V-ATPase H. The dsRNA treatment resulted in a significant reduction in transcript level of targeting gene compared to the control group at 24 h (t=–4.429, df = 4, P= 0.011) and 48 h (t=–3.683, df = 4, P= 0.021) posttreatment; however, the expression level of targeting gene recovered at 72 h (t = 0.583, df = 4, P = 0.591) posttreatment did not differ significantly from that of the control level (Fig. 2).

Fig. 2

Silencing efficiency of double-stranded RNA (dsRNA) treatment in M. alternatus larvae hemolymph. (Error bars are SE. An asterisk [*] shows significantly different between dsRNA targeting vacuolar ATPase subunit H [V-ATPase H] [treatment] and dsRNA targeting enhanced green fluorescent protein [EGFP] [control] at P < 0.05 by pairwise t test.)

Fig. 2

Silencing efficiency of double-stranded RNA (dsRNA) treatment in M. alternatus larvae hemolymph. (Error bars are SE. An asterisk [*] shows significantly different between dsRNA targeting vacuolar ATPase subunit H [V-ATPase H] [treatment] and dsRNA targeting enhanced green fluorescent protein [EGFP] [control] at P < 0.05 by pairwise t test.)

Close modal

Immunity-related gene expression after dsRNA treatment. The expression of immunity-related genes (e.g., lysozyme, TAK1, pelle) was detected at 72 h post–dsRNA treatment. The results indicated that RNAi knockdown of V-ATPase H inhibited the expression of lysozyme (t = –3.658, df = 4, P = 0.022), TAK1 (t = –3.397, df = 4, P = 0.027), and pelle (t = –2.841, df = 4, P = 0.047) (Fig. 3).

Fig. 3

Relative expression of immunity-related gene after double-stranded RNA (dsRNA) treatment in M. alternatus larvae hemolymph. (Error bars are SE. An asterisk [*] shows significantly different between dsRNA targeting vacuolar ATPase subunit H [V-ATPase H] [treatment] and dsRNA targeting enhanced green fluorescent protein [EGFP] [control] at P < 0.05 by pairwise t test.)

Fig. 3

Relative expression of immunity-related gene after double-stranded RNA (dsRNA) treatment in M. alternatus larvae hemolymph. (Error bars are SE. An asterisk [*] shows significantly different between dsRNA targeting vacuolar ATPase subunit H [V-ATPase H] [treatment] and dsRNA targeting enhanced green fluorescent protein [EGFP] [control] at P < 0.05 by pairwise t test.)

Close modal

Vacuolar ATPases are ATP-dependent proton pumps that are involved in various normal cellular processes and disease processes (Jefferies et al. 2008). V-ATPases are widely present in epithelial tissues, such as Malpighian tubules, salivary glands, labial glands, and midgut and sensory sensilla in insects (Wieczorek et al. 2009). It is especially noteworthy that V-ATPases play important roles in the absorption of amino acids and the regulation of pH value in the midgut of insects (Wieczorek et al. 2000). Previous studies show that oral take of dsRNA/siRNA targeting ATPases results in significant mortality as compared with the control in other insect pests (e.g., Diabrotica virgifera virgifera LeConte, Bemisia tabaci (Gennadius), Peregrinus maidis (Ashmead)) (Baum et al. 2007, Upadhyay et al. 2011, Yao et al. 2013). V-ATPase H was highly expressed in the gut in M. alternatus larvae, which provides the possibility of delivery of ds/siRNA orally.

The results from the present study indicate that exogenous dsRNA-induced RNAi efficiency is a transient effect, since the dsRNA triggered a significant reduction of target gene expression at 24 h posttreatment, which recovered at 72 h posttreatment. Fortunately, in planta expression system which produces dsRNA continuously could address this obstacle. For example, transgenic corn (Zea mays Linn) plants expressing rootworm (Diabrotica virgifera virgifera LeConte) dsRNAs decreased rootworm feeding damage significantly in growth chamber assays (Baum et al. 2007), thus illustrating that RNAi pathway can be exploited to control coleopteran insect pests via in planta expression of a dsRNA. In the future, engineering transgenic pine tree expressing M. alternatus dsRNAs may reduce the population burden of this coleopteran insect pest and, in turn, reduce the potential for pine wilt disease spread.

The innate immune system of insects relying on hemolymph defends themselves against infectious organisms (Strand 2008). Antimicrobial peptide lysozyme plays a critical role in insect defense against microbial challenge by cleaving the β-1,4-glycosidic linkage of chitooligosaccharides in fungal cell walls (Kong et al. 2016). TAK1 is required for the immune deficiency signaling pathway in Drosophila. TAK1 mutant fruit flies do not produce antimicrobial peptides upon Gram-negative bacterial infection (Vidal et al. 2001). Pelle, a serine-threonine protein kinase, is an important component of Toll immune signaling pathway that was shown to be essential for induction of antifungal peptide and resistance to fungal infection (Miyasaka and Takatsu 2016). The results from this study showed that RNAi knockdown of V-ATPase H inhibited the expression of lysozyme, TAK1, and pelle, which further confirm the potential of V-ATPase H to be developed as an RNAi target gene for the control of pine sawyer beetle M. alternatus and probably other insect pests.

RNAi is a posttranscriptional gene silencing mechanism in eukaryotic cells, which makes it a powerful tool for gene functional studies, as well as a potential next-generation pest management approach (Wang et al. 2018). Injection, feeding, and topical application are the main methods for delivering dsRNA/siRNA to target organisms (Upadhyay et al. 2011). This represents the first report describing in vitro RNAi experiment in insect hemolymph, which provides a novel environment for evaluating RNAi in insects, especially coleopteran and lepidopteran larvae which contain ample amounts of hemolymph.

This research was supported by the Anhui Provincial Key R&D Program (Grant Number 202004a06020001).

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