Interference with Fatty Acyl-CoA Reductase Reduces Wax Secretion and Influences the Development of Male Ericerus pela (Hemiptera: Coccoidae) Nymphs1
Ericerus pela Chavannes (Hemiptera: Coccoidae) is a typical scale insect that can secrete a waxy covering over its body to protect itself from adverse factors. This insect significantly damages glossy privets and ash trees in most regions of China. The fatty acyl-CoA reductase (FAR) gene is a crucial enzyme in the cutin, suberine, and wax biosynthesis pathways in these insects and also in fatty acid metabolism. Herein, a synthesized double-stranded RNA of the FAR gene in E. pela (dsEpFAR) was used to explore the effects of dsEpFAR on the expression of EpFAR and secretion of wax and development of E. pela. The results showed that a high concentration of dsEpFAR could cause mortality of male nymphs. After a male nymph was treated with a low dose of dsEpFAR, the transcription level of EpFAR was significantly downregulated, the expression level of EpFAR protein was reduced, and the quantity of wax was markedly decreased, suggesting that EpFAR is a key gene in the biosynthesis of wax and that dsEpFAR can interfere with the gene EpFAR and disrupt the process of wax secretion. The growth and development of male nymphs were also significantly influenced by dsEpFAR treatment, leading to a significantly decreased weight and earlier nymph-to-pupae transition. The findings enhance the molecular understanding of male E. pela wax secretion and facilitate the development of EpFAR-based interference with wax secretion for the integrated management of male E. pela.Abstract
The white wax insect Ericerus pela Chavannes (Hemiptera: Coccoidae) commonly occurs in southwestern China (Fig. 1). The male nymphs have no sclerotized cuticle and secrete wax, whereas the females have a hard cuticle and cannot secrete wax. The males comprise 90% of a population. As second-instar nymphs, males move from host plant leaves onto branches where they aggregate and begin to secrete wax that forms a confluent, uniform protective wax layer. Most females in the aggregation disperse individually and not as a group (Chen 2011). In much of China, except southwestern China, this scale insect severely damages glossy privet shrubs and ash trees, leading to heavy economic losses as well as reduction in ornamental value and ecological function of the host plants (Wang and Wang 2014, Zhang and Guo 2018). Ericerus pela is managed primarily by the application of chemical insecticides. Because insecticides are known to harm nontarget species and contaminate the environment, it is necessary to explore new species-specific agents as control methods (e.g., gene or gene products). Furthermore, because the wax secretions serve as a protective shield for male E. pela from insecticide applications, unveiling the function of genes associated with the biosynthesis of wax will be helpful in developing new management strategies for E. pela.
Citation: Journal of Entomological Science 60, 3; 10.18474/JES24-59
RNA interference (RNAi) is an effective posttranscriptional gene-silencing mechanism in eukaryotes and a powerful reverse genetic tool for functional genomic studies (Bellés 2010, Bosher and Labouesse 2000, Jain et al. 2020). Double-stranded RNA (dsRNA) is absorbed through the somatic or midgut cells of insects and processed into small interfering RNAs (siRNAs) by the endoribonuclease Dicer. The siRNAs then bind the complementary mRNA to interfere with the translation process of the target gene (Palli 2014, Wynant et al. 2014). Several studies have uncovered some roles of the target genes by using RNAi and confirmed that RNAi-mediated pest control is a feasible and effective strategy for crop protection, with potential safety and specificity advantages (Dang et al. 2022, Tian et al. 2015, Wang et al. 2021a). For example, the genes AsFAR in Adelphocoris suturalis (Jakovlev) (Hemiptera: Miridae), JHAMT in Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae), and IAP and SNF7 in Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae), which play major roles in the growth, development, or reproduction of these pests, have been confirmed and the introduction of homologous dsRNA of these genes into the corresponding insect hosts caused some typical phenotypes of specific gene deficiency, such as decreased weight, precocious metamorphosis, changed microstructure of epicuticle layer, malformed shape, and cessation of oocyte development (Dhandapani et al. 2020, Guo et al. 2018, Luo et al. 2017). Our question at the onset of this study was can a dsRNA influence the life cycle of a E. pela nymph or/and the wax biosynthesis of male nymphs?
To date, the molecular mechanism of wax biosynthesis is not fully understood, thereby making it very difficult to manage wax-secreting pests by limiting or destroying the wax. It is known that the biosynthesis of wax primarily occurs in two successive catalytic steps: first, the reduction of fatty acyl-CoA to fatty alcohol and second, the subsequent esterification of fatty acyl-CoA and fatty alcohol (Cheng and Russell 2004, Teerawanichpan and Qiu 2010). The fatty acyl-CoA reductase gene (FAR), which encodes the biosynthesis of fatty alcohols and lipids in animals, plants, and microorganisms, is a key gene during development of insects (Jaspers et al. 2014, Li et al. 2020, Munkajohnpong et al. 2020, Wang et al. 2018, 2021b). Tong et al (2022) reported that FAR in mealybug Phenacoccus solenopsis Tinsley influences wax biosynthesis. Ding et al. (2022) analyzed transcriptome data of E. pela and concluded that FAR genes are important catalytic enzymes of fatty acid metabolism. Our previous study (Hu et al. 2018) on E. pela FAR (EpFAR) revealed that EpFAR is highly expressed in the testes and wax glands of male nymphs and can catalyze the reduction of fatty acyl-CoA into fatty alcohol. Because the testis is unique to males and only male E. pela can secrete wax, we hypothesized that EpFAR is key to the development of male nymphs and potentially involved in the biosynthesis of wax.
In this study, a double-stranded EpFAR (dsEpFAR) was synthesized and used to interfere with EpFAR in male E. pela nymphs to identify a potential target gene and illustrate whether the silencing of the target gene can harm normal life processes of E. pela nymphs. Our findings demonstrated that EpFAR plays an essential role in the wax secretion and development of male E. pela nymphs and that dsEpFAR can lead to higher mortality when the concentration of dsEpFAR is high and to a developmental defect with a lower concentration. Therefore, EpFAR is an ideal candidate gene for future development as RNAi-mediated control of E. pela.
Materials and Methods
Insects
Female E. pela adults carrying masses of eggs were collected from glossy privet in a park in Luoyang City (longitude E 112°26′; latitude N 34°37′), China, and held in a shady dry environment. These female adults were assigned to different batches and placed in gauze bags. Next, these bags were hung on the branches of Ligustrum lucidum Ait., which would serve as the hosts for the E. pela nymphs. These hosts were grown under shade in an experimental field at the College of Forestry, Henan University of Science and Technology, Luoyang, China. Newly molted male nymphs, aged 1–2 d, that had moved onto L. lucidum branches and gathered into a group but had not begun to secrete wax were treated with reagent. Because individual nymphs measure only 0.77 mm long and 0.43 mm wide and immobilize themselves on the branch, we selected the coating method to treat them with the reagent.
Synthesis of dsEpFAR
The Simple Modular Architecture Research Tool (http://smart.embl.de/) was used to predict the functional domains of EpFAR. Gene-specific primers for the synthesis of dsEpFAR were designed using Primer Premier 6.0 software based on the conserved sequence and functional domains of EpFAR (Fig. 2A). The plasmid containing the EpFAR open reading frame prepared by Hu et al. (2018) was used as the template to amplify a 759-bp fragment by using gene-specific primers (Table 1). The amplification reactions were performed under the following conditions: predenaturation at 94°C for 4 min, followed by 32 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min by using a CFX96 polymerase chain reaction (PCR) system (Bio-Rad, Hercules, CA). The amplicons were purified using a PCR product recovery kit (SunShine Bio, Ltd., Shanghai, China). Next, approximately 1 μg of the recovered DNA per tube was used as the template to generate dsEpFAR by using a MEGAscript® RNAi kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Finally, the concentration of dsEpFAR was measured by using a Thermo ND2000 spectrometer (Thermo Fisher Scientific, Waltham, MA).
Citation: Journal of Entomological Science 60, 3; 10.18474/JES24-59
Nontoxic dose of the surfactant screening
Triton X-100 (Sigma-Aldrich, St. Louis, MO) was used as a surfactant to make the solution of dsEpFAR uniformly adhere to the body of a nymph to facilitate dsEpFAR penetration through the cuticle. An effective and nontoxic dose of surfactant was screened first according our experimental program. Newly molted male nymphs on L. lucidum branches were treated with 3, 1, 0.3, 0.1, and 0.03% Triton X-100. Triton X-100 was coated on the body of nymphs by using a small piece of soft cotton. Next, double-sided adhesive tape was wrapped around the base of branches to inhibit untreated nymphs from joining the treated group; a fine mesh was used to shield the treated group from parasites or predators (Fig. 2B). The controls were treated with sterile water. The assay was conducted in three replicates. Each replicate of ≥30 nymphs was based on a group of male nymphs, given the gregarious life of male E. pela. The toxicity of Triton X-100 was assessed 5 d after treatment by determining the number of dead nymphs. Dead nymphs were identified based on color changes and detachment from their inhabiting sites when brushed with a soft hairbrush. A dose that caused no significantly different mortality from the control, and with excellent adhesion ability, was deemed as a nontoxic dose and used in follow-up assays.
Tolerated dose of dsEpFAR screening
To determine whether the dsEpFAR could lead to mortality, we investigated the effects of dsEpFAR on the lives of healthy nymphs. Triton X-100 is a nonionic detergent with the ability to improve the permeability of eukaryotic cell membrane in biological experiments, reduce the surface tension of dsEpFAR solution, and help dsEpFAR quickly adhere to the nymphal epidermis so as to be easily taken up through endocytosis. Based on the results from the aforementioned assay, a nontoxic dose of Triton X-100 was used as the surfactant. The concentrations of dsEpFAR used in the experiment were 10, 20, 40, 80, and 160 ng/µl. The surfactant was coated first, followed by dsEpFAR, as described in the previous section. Groups treated with dsEGFP (Zsyg Biotech Co. Ltd., Shanghai, China) instead of dsEpFAR served as the controls. The assay was performed in three replicates. The tape and mesh were also used as protection, and the lethal effect of dsEpFAR was assessed using the method described in the previous section. The dose that caused no significantly different mortality from the control was deemed as a tolerated dose of dsEpFAR and was used in the RNAi assay.
RNAi assay
We performed an RNAi assay by using a tolerated dose of dsEpFAR to explore the impacts of dsEpFAR on the expression of EpFAR and the development of nymphs. Newly molted second-instar nymphs, which had not yet begun to secrete waxy secretion, were used in these assays. Coating dsEpFAR solution on the body was selected and used after comparing and analyzing the spraying and soaking methods (Maeda et al. 2001, Tabara et al. 1998, Wang et al. 2011, Yan et al. 2020). Triton X-100 (nontoxic dose) and dsEpFAR (tolerated dose) were coated as described in the previous two sections. The control groups were treated with dsEGFP replacing the dsEpFAR solution. In total, 44 groups were assigned to three treatments (each with 22 groups) and treated with dsEpFAR or dsEGFP. Double-sided adhesive tape and fine mesh were used for protective measures as described previously (Fig. 2B). From each treatment, 18 of the 22 groups with >30 nymphs per group were divided into three batches that were collected on days 2, 4, and 8 posttreatment and preserved in liquid nitrogen for the subsequent extraction of total RNA or protein. Twenty nymphs were collected from each group. The other eight groups (four groups in the dsEpFAR and four in the control treatments) with >60 nymphs per group were allowed to continue infesting the hosts, and their phenotypic characterizations, including wax secretion, weight gain, and pupation, were calculated at the end of the experiment.
Analysis of efficiency of interference
The level of transcription of EpFAR in nymphs treated with dsEpFAR was determined using a real-time quantitative reverse-transcription PCR (qRT-PCR). Total RNA was extracted from the whole insect body by using an EZ-10 DNAaway RNA mini-prep kit (Sangon Biotech, Shanghai, China) following the manufacturer’s instructions. Next, 1 µg of RNA was reverse transcribed using an RT EasyTM II kit (Foregene, Chengdu, China) according to the manufacturer’s instructions. Gene-specific primers for qRT-PCR were designed using Primer Premier 6.0 and are listed in Table 1. The qRT-PCR was performed using a CFX96 real-time PCR system (Bio-Rad) in a 20-µl reaction mixture that contained 10 µl of Universal SYBR qPCR master mix (Biosharp, Shanghai, China), 20 ng of synthesized cDNA, and 0.2 µM of forward and reverse primers. The reaction was performed under the following conditions: predenaturation at 95°C for 2 min, followed by 39 cycles of denaturation at 95°C for 15 s and annealing at 60°C for 30 s. β-Actin was used to normalize the gene expression. The experiment was conducted in three replicates. The relative level of expression of EpFAR was analyzed using the 2−ΔΔCt method (Schmittgen and Livak 2008).
To investigate the effects on the EpFAR protein after the dsEpFAR treatment, changes in the EpFAR protein were analyzed by western blotting. The nymphs treated with dsEpFAR were collected on days 2, 4, and 8 after treatment. Their total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China) following the manufacturer’s instructions. The concentration of EpFAR was determined using an enhanced BCA protein assay kit (Beyotime). Equal proteins from different samples were fractionated by electrophoresis by using a 12% sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The fractionated proteins were transferred onto nitrocellulose membranes and immunolabeled at 37°C for 1 h with a rabbit polyclonal anti-EpFAR antibody (1:200) as described previously (Hu et al. 2014) and incubated with horseradish peroxidase–coupled secondary antibody (1:5,000, Zoonbio Biotechnology Co., Ltd., Nanjing, China). Immunolabeling was detected using an electrogenerated chemiluminescence western blotting system (Roche, Diagnostics, Shanghai, China), with β-actin (1:5,000) as the loading control.
Phenotypic characterization of E. pela after RNAi
The characteristics of the secreted wax were assessed using a VHX-5000 microscopic imaging system (Keyence, Osaka, Japan) to investigate the effect of dsEpFAR on the ability of E. pela to secrete wax. Investigations were on days 2, 4, and 8 after treatment with dsEpFAR. To assess the cumulative impact of dsEpFAR on the potential to secrete wax and the development of E. pela, the cumulative quantity of wax, weight of the nymph, and metamorphosis of nymphs at the end of the assay were determined when the first male adult emerged in the treatment or control groups. The numbers of molted male adults trapped in each mesh were counted. The waxy covering with nymphs and pupae was detached from the branches. All the nymphs and pupae in the waxy covering were removed using smooth tweezers. Next, the nymphs, propupae, true-pupae (True-pupa, a real pupa, its wing, leg and antennae are visible. true-pupa is different from propupa which is similar to its nymphal shape.), and male adults were separated and counted. The waxy covering and nymphs in each group were separately weighed using a high-precision balance (Mettler Toledo, Shanghai, China). This assay was replicated four times with a single group serving as a replicate.
Statistical analysis
The data were analyzed using SPSS 20.0 (IBM, Inc., Armonk, NY). For the toxicity of surfactant and dsEpFAR, the percent mortality was corrected by adjusting the treatment mortality with that of the control using Abbott’s correction (Abbott 1925). The corrected mortality (Rcr) was calculated using the following formula: Rcr (%) = (Rtr − Rco)/(1 − Rco) × 100%, where Rtr is the mortality rate of the group treated with dsEpFAR and Rco is the mortality rate of the control group. The differences in mortality were analyzed using a one-way analysis of variance, and the means were separated by a Tukey’s honestly significant difference multiple comparison test (α = 0.05). Before analysis, the variance was normalized by arcsine-square root transformation of the percentage data (Yang et al. 2020). To assess the phenotypic changes, the pupation rate (Ppu), the proportion of propupa (Ppr), and the proportion of true-pupa (Ptr) per group were calculated using the following formulae: Ppu (%) = (Npr + Ntr + Nad)/(Nla + Npr + Ntr + Nad) × 100%; Ppr (%) = Npr/(Nla + Npr + Ntr + Nad) × 100%, and; Ptr (%) = (Ntr + Nad)/(Nla + Npr + Ntr + Nad) × 100%, where Nla, Npr, Ntr, and Nad represent the number of nymphs, propupae, true-pupae, and adults in a group, respectively. To determine the weight of wax secreted per nymph (Wwa) and the weight per nymph (Wla), the following formulae were used: Wwa = Wwq/(Nla + Npr +Ntr + Nad) and Wla = Wtl/Nla, where Wwq represents the weight of wax covering secreted per group and Wtl represents the weight of nymphs in a group. The differences in the levels of expression of EpFAR, wax weights, nymphal weights, pupation rate, or the proportion of propupa and true-pupa between the study control groups compared using a Student’s t test (P < 0.05). Before analysis, the rate and proportion data were transformed by arcsin √x.
Results
Nontoxic dose of surfactant
The mortality of nymphs treated with different concentrations of Triton X-100 is shown in Fig. 3A. The corrected mortality rate of the nymphs coated with 3, 1, 0.3, 0.1, and 0.03% Triton X-100 was 8.1, 3.22, 2.23, 1.48, and 0%, respectively. There was a significant difference in the mortality rates between the groups treated with Triton X-100 and the control (F = 4.260; df = 5, 12; P = 0.018). However, when the concentration of Triton X-100 was <1%, the difference between the mortality of the groups treated with Triton X-100 and the control was insignificant, suggesting that the nymphal life was not affected by Triton X-100 when the concentration of Triton X-100 was <1%. Specifically, 0.1% Triton X-100 had negligible adverse effects on the lives of E. pela nymphs and good affinity; thus, it was deemed as the nontoxic dose and used as the surfactant during dsEpFAR toxicity and RNAi assays.
Citation: Journal of Entomological Science 60, 3; 10.18474/JES24-59
Tolerated dose of dsEpFAR
The dsEpFAR toxicity assay revealed that 160 and 80 ng/µl dsEpFAR caused a relatively higher rate of nymphal mortality (37.79 and 11.76%, respectively), which was significantly different from the control (Fig. 3B; F = 21.617; df = 5, 12; P < 0.0001). However, the differences in the rates of nymphal mortality between the dsEpFAR and the control groups were insignificant when the dsEpFAR concentrations were ≤40 ng/µl. In particular, the mortality rate at 40, 20, 10 and 0 ng/µl (control) dsEpFAR was 4.58, 3.38, 2.91, and 4.39%, respectively. Therefore, 40 ng/µl dsEpFAR was deemed as the tolerated dose and used in the RNAi assay to establish the relationship between EpFAR and wax secretion.
Expression of EpFAR in nymphs treated with dsEpFAR
The effect of dsEpFAR on EpFAR in E. pela nymph is shown in Fig. 4. The relative transcription level of EpFAR in the second-instar nymphs treated with dsEpFAR was significantly downregulated on days 2, 4, and 8 compared with the control (day 2: t = 18.687; df = 4; P < 0.0001; day 4: t = 13.727; df = 4; P < 0.0001; and day 8: t = 4.512; df = 4; P = 0.011). On days 2, 4, and 8, the expression of EpFAR was reduced by 4.69-, 2.45-, and 1.50-fold after treatment with dsEpFAR, respectively. The results suggest that the dsEpFAR can disrupt the transcript of gene EpFAR in E. pela nymph.
Citation: Journal of Entomological Science 60, 3; 10.18474/JES24-59
DsEpFAR influence on accumulation of EpFAR protein
The effect of dsEpFAR on the EpFAR protein in E. pela nymph based on western blotting is summarized in Fig. 5. The EpFAR protein in the groups treated with dsEpFAR and the control clearly differed. The amount of EpFAR protein in the nymph treated with dsEpFAR was very low on days 2 and 4 after treatment. On 8 d posttreatment, the quantity of EpFAR protein in the group treated with dsEpFAR increased compared with that of days 2 and 4. However, the amount of EpFAR protein in the control group on days 2, 4, and 8 did not clearly vary. The level of EpFAR protein showed the variation tendency of rising after falling with time due to dsEpFAR treatment. The variation trend of the EpFAR protein was consistent with the change in the level of transcription of EpFAR in the nymphs treated with dsEpFAR. The results suggest that dsEpFAR can disrupt the expression of EpFAR protein.
Citation: Journal of Entomological Science 60, 3; 10.18474/JES24-59
Wax secretion capability after dsEpFAR treatment
To evaluate the influence of RNAi on wax secretion, the changes in wax secretion were recorded at different times and the cumulative wax during the assay period was also evaluated. On day 2 posttreatment, the wax secretion feature of the nymphs treated with dsEpFAR was similar to that of the control, with only very little wax “powder” on the nymphal body surface (Fig. 6). On 4 d posttreatment, a little wax filament was sparsely scattered on the nymphal body surface and a clump of wax filament on the side of the thorax was visible in the group treated with dsEpFAR. By contrast, a layer of wax filament covered the backs of nymphs in the dsEGFP-treated group, which obscured the the nymphal body. On day 8 posttreatment, the nymphs in the groups treated with dsEpFAR remained visible, although there was a slight increase in the wax filament compared with those at 4 d. However, a very dense layer of wax filament, named wax quilt, had entirely covered the nymphs in the dsEGFP-treated group on day 8 posttreatment. The results suggest that the nymphs cannot normally secrete wax after dsEpFAR treatment.
Citation: Journal of Entomological Science 60, 3; 10.18474/JES24-59
The cumulative wax was collected and weighed at the end of the RNAi assay. The maximum weight of fresh wax secreted by each nymph in the dsEpFAR treatment group was 0.359 mg/♂ and the minimum weight was 0.305 mg/♂, with an average weight of 0.341 mg/♂, which was significantly lower than that of the control with an average weight of 0.409 mg/♂ (t = 2.458; df = 6; P = 0.049). These results imply a weakened capability of wax secretion for the dsEpFAR-treated nymphs (Fig. 7).
Citation: Journal of Entomological Science 60, 3; 10.18474/JES24-59
Effects of dsEpFAR on growth and development of E. pela
To investigate the effects of EpFAR interference on the growth and development of E. pela, the weight of nymphs was examined at the end of the RNAi assay. The male nymphs of E. pela treated with dsEpFAR weighed 0.28 mg/nymph, on average, whereas the control weight was 0.32 mg (Fig. 8A), which was significantly different (t = 2.806; df = 6; P = 0.031). The results suggest that dsEpFAR inhibited the growth of male nymphs.
Citation: Journal of Entomological Science 60, 3; 10.18474/JES24-59
Interestingly, the uptake of dsEpFAR significantly affected the rate of nymphal pupation with 24% of those treated with dsEpFAR (t = 5.522; df = 6; P = 0.001). However, in the control group, the pupation rate was 5.7% where most individuals were in the immature stage and secreting wax. Furthermore, the rate of emergence of true-pupae from the groups treated with dsEpFAR was higher than that of the control (t = 4.348; df = 6; P = 0.005). In particular, the ratio of true-pupae to total pupae in the group treated with dsEpFAR was 3.16-fold higher than that in the dsEGFP-treated group (Fig. 8B). Indeed, in the process of experiment, the first male adult emerged from the group treated with dsEpFAR, and no male adult was observed in the dsEGFP-treated group until the end of the assay. The significant difference in the pupation rate showed that the nymphs treated with dsEpFAR entered the pupal stage earlier than what is customary, suggesting the dsEpFAR affected the development of the nymphs. From another point of view, the early pupation implied that the period of feeding damage in the life of male E. pela was clearly shortened compared with that of the control.
Discussion
Ericerus pela is a forestry pest in most regions of China, except southwestern China. The broad-leaved forest and urban green land with glossy privets and ash trees as the main tree species are severely damaged by this pest. The affected glossy privets and ash trees are covered with white wax that seriously affects photosynthesis and causes tree mortality. The male nymphs have no sclerotized cuticle and are highly dependent on the waxy covering to protect them from harmful biotic and abiotic factors. Interrupting the synthesis of wax presented an intriguing approach to the management of E. pela. It was confirmed that dsRNA can kill the pest based on destroying the function of target gene, and its success greatly depends on suitable target genes (Dhandapani et al. 2020, Luo et al. 2017, Wang et al. 2021a). RNAi also can affect the development of pests because some loss-of-function phenotypes were generated through a depletion of chosen transcript (Bellés 2010, Jain et al. 2020). In this study, by using the RNAi technology, we unveiled the role of EpFAR, with the results showing promise for RNAi-based E. pela management.
FARs are the key enzymes in the biosynthesis of fatty alcohols, which are precursors of wax esters, cuticular hydrocarbons, and sex pheromones in some insects (Dou et al. 2020, Finet et al. 2019, Li et al. 2019). For E. pela, the downregulation of EpFAR conspicuously decreased the wax secretion within the first 8 d, followed by a significant decrease in the cumulative wax at the end of the trial. This suggested that EpFAR is essential for the biosynthesis of wax. The wax, as is known to all, has a function as a protective barrier for insects. So, once the wax on the surface of the body of E. pela decreases due to dsEpFAR treatment, E. pela probably loses an effective protective barrier that is potentially fatal to E. pela. During the process of experiment, there appeared to be a slight increase in the relative expression level of EpFAR after day 8 posttreatment, which might be induced by the degradation of dsEpFAR due to some unknown factors. Regardless, there was EpFAR interference and the secretion of wax was decreased, which was helpful for further research of dsEpFAR.
Larval weight is generally a health indicator, and weight loss is associated with abnormal larval development. Luo et al. (2017) reported a series of changes in female A. suturalis following the downregulation of AsFAR at day 0 posteclosion, including a significant decline in the number of oocytes, dry weight of ovaries, lifetime fecundity, and egg hatchability. Adult weight and number of eggs laid by L. decemlineata adults fed on plant-derived dsRNA as larvae were decreased (Guo et al. 2018). In our study, the fresh nymphal weight in the group treated with dsEpFAR significantly decreased, suggesting that the nutrition metabolism of E. pela was likely interrupted by dsEpFAR. Therefore, it can be concluded that EpFAR is related to the development and growth of E. pela and that dsEpFAR is a severe threat to the nymphal health.
In this study, dsEpFAR accelerated the metamorphosis of E. pela nymphs: many treated nymphs entered the pupal stage earlier than those in the natural process. In addition, the proportion of true-pupa in the group treated with dsEpFAR was higher than that in the control, implying that pupation in the group treated with dsEpFAR took place earlier than in the control. The precocious metamorphosis suggested that the developmental duration of male nymphs was shortened, possibly due to their abnormal growth and development. Phenotypic changes under gene deficiency induced by dsRNA have been observed in other insects, such as precocious metamorphosis in Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and L. decemlineata (Guo et al. 2018, Minakuchi et al. 2008); smaller basal oocytes in Schistocerca gregaria Forsskål (Orthoptera: Acrididae) induced by the knockdown of specific genes (Marchal et al. 2011); malformed oocytes caused by dsNlFAR4, dsNlFAR6, and dsNlFAR9 in the brown planthopper (Nilaparvata lugens (Stål)) (Li et al. 2020); and altered microstructure of the epicuticle layer due to the knockdown of NlFAR in N. lugens (Li et al. 2019). In addition, the available evidence also indicated that typical phenotypes of specific gene deficiency and sensitivity to RNAi are different among insect species (Christiaens et al. 2014, Coleman et al. 2015, Zhang et al. 2015). In this study, the E. pela nymphs pupated earlier because of dsEpFAR, which shortened the period of damage to the host. Because phenotypic abnormality significantly influenced individual vitality, successful interference with EpFAR will effectively lower population viability.
An important challenge for most insect RNAi studies, especially for the future application of dsRNA for pest control practice, is developing a delivery method. Thus far, two dsRNA application methods are being developed: host-induced gene silencing wherein the host plant is engineered to express the dsRNA molecules and spray-induced gene silencing (SIGS) that avoids the challenges surrounding the regulation of genetic modification (Wytinck et al. 2020). SIGS using naked dsRNA has been shown to be an effective control method against some insect pests (Killiny et al. 2014, Zhang et al. 2020). Among the SIGS methods, the soaking method is a classical method successfully applied to the nematode Caenorhabditis elegans (Maupas) (Tabara et al. 1998). The epidermal microapplication of dsRNA also successfully induced interference of the target genes in the Asian citrus psyllid Diaphorina citri Kuwayama (Hemiptera: Liviidae) (El-Shesheny et al. 2013). In some reports, nanoparticles, cell-penetrating peptides, or cationic liposomes were applied in the laboratory or greenhouse environment with the goal of decreasing the degradation of dsRNA, although they can partly increase the possibility of dsRNA delivery (Adams et al. 2019, Chen et al. 2012, Joga et al. 2016, Lin et al. 2017, Milletti 2012, Zhou et al. 2015). However, the production costs of these dsRNA carriers and the economic cost of RNAi-based technologies exceed levels deemed appropriate for the wide-scale field use (Zotti et al. 2018). In our study, these newly molted nymphs measure only 0.78 × 0.43 mm and are fixed on host plant branches, both factors limiting application by injection. We found that the epidermal application of dsRNA by using the soaking methods coated the bodies of newly molted nymphs with the dsEpFAR solution. We also found that this coating must remain in place for 60 s for the dsEpFAR to infiltrate the epidermis as much as possible. At this point, the newly molted nymphs have not yet begun to secrete waxy secretion and they are likely more sensitive to the dsRNA than those at older stages of development. Furthermore, wax glands are present on nymph epidermis and it has been confirmed that the gene EpFAR is expressed mainly in wax glands (Hu et al. 2018).
Pest management of scale insects typically targets females, which contrasts with the management of E. pela where males are the key targets. The fecundity of female E. pela is naturally high, with the female-to-male ratio being 1:4. The male nymphs comprise a very large percentage of the total E. pela population; therefore, limiting the number of male E. pela could significantly reduce the damage they cause to infested plants. Nevertheless, given the relationship between population and female fertility, it is necessary to explore the role of EpFAR in female E. pela in the future.
Male Ericerus pela endangering glossy privet.
Schematic illustration of the RNAi region (73–831 bp) in the conserved sequence and functional domains of the Ericerus pela fatty acyl-CoA reductase gene (A) and the method of treating the nymphs in this study (B).
Lethal effect following treatment with Triton X-100 (A) and dsEpFAR (B). The vertical lines represent the SEM mortality rates (n = 3), whereas the asterisks indicate significant differences in the mortality rates between the groups treated with dsEpFAR and those of the control (analysis of variance, Tukey’s honestly significant difference test, P < 0.05).
Relative expression (fold) of EpFAR in the male nymphs at days 2, 4, and 8 after treatment with dsEpFAR. The error bars indicate the SEM (n = 3), whereas the asterisks indicate statistically significant differences between the groups treated with dsEpFAR and those of the control (Student’s t test; ∗P < 0.05; ∗∗P < 0.01).
Effects of dsEpFAR on the EpFAR protein in male nymphs at 2, 4, and 8 days posttreatment.
Wax secretion of Ericerus pela at days 2, 4, and 8 after treatment with dsEpFAR. 2d: amount of wax both in dsEpFAR group and dsEGFP group is comparable; 4d: a little wax filament was visible in dsEpFAR group, but in dsEGFP group, there was a layer of wax filament; 8d: some wax filament is on the body surface in dsEpFAR group, but in dsEGFP group, there was so much wax that the body of nymph was invisible.
Effects of dsEpFAR on the cumulative wax per nymph. The error bars represent the SEM (n = 4), whereas the asterisks indicate statistically significant differences between the groups treated with dsEpFAR and the control groups (Student’s t test; ∗P < 0.05).
Effects of dsEpFAR on the nymphal growth and development. (A) Net weight gain per nymph. (B) Pupation rate and the proportion of propupa and true-pupa. The error bars represent the SEM (n = 4), whereas the asterisks indicate statistically significant differences between the groups treated with dsEpFAR and the control groups (Student’s t test; ∗P < 0.05; ∗∗P < 0.01).
Contributor Notes