Insect rearing
The cotton mealybugs used in this study were originally collected from Rose of Sharon, Hibiscus syriacus L. (Malvales: Malvaceae) in Jinhua, Zhejiang Province, China, in June 2016. They were maintained on fresh tomato plants (cv. Hezuo-903, Shanghai Changzhong Seeds Industry Co., Ltd, China) in a climatically controlled chamber maintained at 27 ± 1 °C, 75% relative humidity (RH), and a photoperiod of 14:10 (L:D). For detailed insect rearing and tomato cultivation methods see ref. 56.
Scanning electron microscopy (SEM) of P. solenopsis wax
SEM was used to observe changes in wax on the body surface of adult P. solenopsis females according to the methods of Huang et al.57. Briefly, collected insects were taped onto a stub and dried in an ion sputter (Hatachi, Tokyo, Japan) under a vacuum. After gold sputtering, the samples were observed using a TM-1000 SEM (Hatachi, Tokyo, Japan). Photos were scanned from the dorsal part of the third thoracic segment. Thirty insects were used for both RNAi-treated and control groups.
Chemical composition analysis of mealybug wax
A small soft brush was used to collect wax filaments from the body surface of P. solenopsis females. Prior to use, the brush was washed successively by 70% ethanol, sterile water, and 1× sterile phosphate-buffered saline (PBS, pH 7.4). The wax was collected into a clean chromatography vial for the following experiments. Two vials of wax, each collected from 1000 adult females, were dissolved in 1 ml of methanol and 1 ml of n-hexane, respectively. The vials were stirred gently for 3 min, kept at room temperature for 30 min, and then put into an S06H ultrasonic vibrator (Zealway, Xiamen, China) for 30 min to dissolve the wax sufficiently. The samples were analyzed on a TRACE 1310 (Thermo Scientific, Waltham, USA) gas chromatograph (GC) equipped with an ISQ single quadrupole MS and interfaced with the Chromeleon 7.2 data analysis system (Thermo Scientific, Waltham, USA), with a constant flow of helium at 1 ml/min. For each sample, a splitless injection of 1.0 μl was respectively made into a polar TG-WaxMS (Thermo Scientific, Waltham, USA) and a nonpolar TG-5MS (Thermo Scientific, Waltham, USA) 30 m × 0.25 mm × 0.25 μm capillary column. The temperature program for polar column samples was as follows: 40 °C for 2 min, then 5 °C/min to 240 °C, hold 10 min; the program for nonpolar column samples was: 40 °C for 2 min, then 5 °C/min to 300 °C, hold 5 min. Injector and detector temperatures were, respectively, set at 250 and 230 °C for polar column samples, and at 300 and 300 °C for nonpolar column samples. Mass detection for all samples was run under an EI mode with a 70 eV ionization potential and an effective m/z range of 35–450 at a scan rate of 5 scan/s. Chemical compounds were identified by mapping against the NIST database. The relative content of each compound was calculated by peak area which was determined using the Agilent MassHunter system.
RNA extraction and RT-qPCR
Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions, and RNA quality was accessed using agarose gel electrophoresis and a Biodrop μLite. 800 ng of total RNA was used for cDNA synthesis using the HiScript III RT SuperMixfor qPCR (+gDNA wiper) (Vazyme Biotech Co., Ltd., Nanjing, China), according to the manufacturer’s instructions. Quantitative RT-PCR (RT-qPCR) was conducted using an AriaMx real-time PCR system (Agilent Technologies, USA), using a 20 μl reaction containing 2 μl of 10-fold diluted cDNA, 0.8 μl of each primer, and 10 μl ChamQ SYBR Color qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China). The RT-qPCR thermocycling protocol was 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The PsActin gene was used as an internal control. At least three biological replicates were used for each experiment. Quantitative variations were evaluated using the relative quantitative method (2−ΔΔCt)58.
Transcriptome analysis of integumentary and non-integumentary tissues
To obtain the integument and other tissues, adult P. solenopsis females were dissected in 1× sterile PBS (pH 7.4) on a sterile Petri dish. Dissected fresh tissues were directly used or frozen in liquid nitrogen and stored at −80 °C for follow-up experiments. We sequenced the transcriptomes of integumentary and non-integumentary tissues (all other tissues without integument) dissected from 150 adult females, with each sample being repeated in triplicate. mRNAs were purified from total RNA via oligo (dT) magnetic beads, and the fragmented mRNAs were then reverse transcribed into cDNA using random primers. Constructed pair-end libraries were sequenced using an Illumina HiSeq X Ten platform in Novogene (Beijing, China). After quality control, the clean RNA-Seq data of the six libraries were aligned with the P. solenopsis genome (http://v2.insect-genome.com/Organism/624) using HISTAT259. Then featureCounts60 and DESeq261 were used for the differential expression analysis of genes. The threshold for differentially expressed genes (DEGs) was defined by log2fold ≥ 1 or ≤−1 and a padj-value < 0.05.
Proteome analysis of the integument
Integuments dissected from 300 adult P. solenopsis females were ground in liquid nitrogen using a mortar and pestle, then dissolved in 400 μl SDT lysis buffer (4% SDS, 100 mM Tris–HCl, 1 mM DTT, pH 7.6). After sonication (ten pulses of 10 s with 10 s intervals, 100 W) and 15 min boiling, samples were centrifuged at 13,000×g at 4 °C for 40 min. Proteins were quantified by the BCA method (Solarbio, Beijing, China). Protein bands were checked by SDS–PAGE and Coomassie blue staining. In total, 300 μg of proteins were used in this experiment. The 100 mM DTT was removed by repeated ultrafiltration (10 kD microsep) using 200 μl of UA buffer (8 M urea, 150 mM Tris–HCl, pH 8.0), then 100 μl of iodoacetamide (100 mM in UA) was added to block reduced cysteine residues, and the samples were incubated for 30 min in the darkness. The filters were washed twice in 100 μl of UA buffer and twice in 100 μl of 25 mM NH4HCO3 buffer. Finally, the protein suspensions were digested with 6 μg trypsin (Promega) in 40 μl of 100 mM NH4HCO3 buffer for 16–18 h at 37 °C. The resulting peptides were desalted on C18 cartridges (Empore SPE Cartridges C18 (standard density), bed ID 7 mm, volume 3 ml) (Sigma-Aldrich, MO, USA), concentrated by vacuum centrifugation, and reconstituted in 40 μl of 0.1% (v/v) formic acid.
LC–MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Fisher Scientific) coupled with Maxquant software (Thermo Fisher Scientific). Here, 2 μg of high pH reserved-phase peptide fragments were loaded onto a reverse-phase trap column (Thermo Fisher Scientific EASY column, 100 μm × 2 cm, 5 μm-C18) connected to the C18-reversed-phase analytical column (Thermo Fisher Scientific Easy Column, 75-μm inner diameter, 10-cm long, 3 μm resin) in buffer A (0.1% formic acid) and separated with a linear gradient of buffer B (0.1% formic acid and 84% acetonitrile) at a flow rate of 300 nl/min. The eluted peptides were ionized, and the full MS spectrum (from m/z 300 to 1800) was acquired by a precursor ion scan using the Q-Exactive analyzer with a resolution of r = 70,000 at m/z 200, followed by a 20 MS2 scan in the Q-Exactivea analyzer with a resolution of r = 17,500 at m/z 200. The MS raw files were translated into mgf files and searched against the integumentary transcriptome using Maxquant 1.3.0.5. Trypsin was defined as the cleavage enzyme allowing no more than two missed cleavages. Carbamidomethylation of cysteine was specified as a fixed modification, and oxidation of methionine was specified as a variable modification. Proteome extraction and sequencing were performed by Applied Protein Technology (Shanghai, China). To verify the existence of corresponding proteins expressed by integument upregulated DEGs, blast+ 2.12.0 was used to blast translated protein sequences of integumentary upregulated DEGs against the integumentary proteome data with the e-value set at 1E-5.
Rapid amplification of cDNA ends of the PsFAR gene
The SMART™ RACE cDNA Amplification Kit (Takara, Kyoto, Japan) was used to obtain full-length cDNAs of the PsFAR gene. 5’-UTR and 3’-UTR RACE cDNAs were synthesized from total RNA using SMARTScribeTM Reverse Transcriptase (Clontech), according to the manufacturer’s instructions. Specific primers for PsFAR (Supplementary Table 6) were designed using Primer-BLAST in NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Paired with the Universal Primer Mix supplied in the kit, one pair of forward and reverse gene-specific primers were, respectively, used in the 3’ and 5’ RACE first-step PCR reactions. PCR conditions were as follows: incubation at 94 °C for 3 min; five cycles at 94 °C for 30 s, 72 °C for 3 min; five cycles at 94 °C for 30 s, 68 °C for 30 s, 72 °C for 3 min; and 25 cycles at 94 °C for 30 s, 66 °C for 30 s, 72 °C for 3 min. The final extension was 72 °C for 10 min. PCR products were purified using the FastPure Gel DNA Extraction Mini Kit (Vazyme Biotech Co., Ltd., Nanjing, China) and cloned using the pClone007 Simple Vector Kit (Tsingke Biotech, Beijing, China). Positive clones were selected and sequenced in Tsingke Biotech.
Sequence analysis of PsFAR
The amino acid sequences of FARs were deduced from the corresponding cDNA sequence using ORFfinder in NCBI (https://www.ncbi.nlm.nih.gov/orffinder/). Multiple sequence alignments were conducted with ClustalX and GeneDoc. The phylogenetic tree was constructed in MEGA X62, wherein neighbor-joining algorithm analysis using the JTT model for amino acids with 1500 bootstrap replicates was performed. Organisms and the GenBank accession numbers of sequences used here are shown in Supplementary Data 3.
dsRNA synthesis and microinjection
dsRNA was synthesized using the T7 High Yield RNA Transcription Kit (Vazyme Biotech Co., Ltd., Nanjing, China) according to the manufacturer’s instructions. Briefly, the DNA template for dsRNA synthesis was amplified with primers containing the T7 RNA polymerase promoter at both 5’ ends (Supplementary Table 6). The purified DNA template (200 ng), a unique 435 bp fragment of PsFAR, was then used as templated for dsRNA production. dsGFP was used as a negative control. Synthesized dsRNAs were purified via isoamyl alcohol precipitation and re-suspended in nuclease-free water, and the concentration was quantified with a UV5NANO (Mettler-Toledo, Zurich, Switzerland). Finally, the quality and size of dsRNAs were further verified via electrophoresis in a 1% agarose gel.
Microinjection of P. solenopsis was conducted with the Eppendorf InjectMan NI 2 microinjection system (Eppendorf, Hamburg, Germany). 4–5-day-old third-instar females were collected and pooled as a single biological replicate. After anaesthetizing with CO2 for 30 s, ~200 ng of dsRNA was injected into the ventral thorax between the mesocoxa and hind coxa. After injection, insects were kept in a plastic rearing box (14 cm long, 10.5 cm wide, 5 cm high) containing one fresh tomato branch. Tomato branches were wrapped with moistened cotton at their base to provide water and renewed every 4 days.
Vector construction and tobacco transformation of dsPsFAR
To express dsPsFAR in tobacco, the 422 bp fragment of PsFAR was first amplified using a forward primer containing BamHI and SalI restriction sites and a reverse primer containing SacI and ApaI restriction sites (Supplementary Table 6). The sense and antisense strands of the purified PsFAR fragment were respectively double digested by SalI + ApaI and SacI + BamHI. Two digested strands were inserted into corresponding restriction sites of the plant expression vector pCAMBIA130163 one at a time. The resulting RNAi transformation vector pCAMBIA1301-dsPsFAR was validated by sequencing (Tsingke Biotech, Beijing, China).
The pCAMBIA1301-dsPsFAR vector was introduced into tobacco (Nicotiana tabacum cv. Petit Havana) via Agrobacterium-mediated transformation. After transformation and culture, leaf discs were washed three times with distilled water and dried on absorbent paper. Then, leaf discs were pre-selected with kanamycin and transferred for differentiation and rooting. Regenerated plantlets were cultivated in a greenhouse for selection. Genetic transformation of tobacco was entrusted to Towin Biotechnology (Wuhan, China).
Validation of dsPsFAR expression in GM tobacco plants
The T5 Direct PCR Kit (Plant) (Tsingke Biotech, Beijing, China) was used to identify positive T0/T1 dsPsFAR genetically modified (GM) tobacco plants. Briefly, leaves 1–2 mm in diameter of T0/T1 GM and wild-type (WT) N. tabacum plants were lysed in 50 μl of lysis buffer A, followed by 10 min of incubation at 95 °C. After fully shocking by hand for 30 s and a brief centrifuge, the supernatant was used for PCR amplification. The 50 μl PCR reaction contained 1 μl of template DNA (i.e. supernatant), 2 μl of each primer and 25 μl of 2×T5 Direct PCR Mix (Plant) and the following thermocycling conditions were used: initial denaturation at 98 °C for 3 min; 35 cycles at 98 °C for 10 s, 63 °C for 10 s, 72 °C for 15 s; and final extension at 72 °C for 5 min. Primers used here are listed in Supplementary Table 6; the RbcL gene in vascular plants was used as a positive control. Genomic DNA from WT plants and double-distilled water were both used as negative controls. PCR products were analyzed by agarose gel electrophoresis.
To select for GM plants with the highest dsPsFAR expression for subsequent bioassays, total RNA of leaves from 9 randomly selected T0 GM tobacco plants (50-day old) exhibiting similar growth were isolated. The relative expression levels of dsPsFAR were quantified by RT-qPCR, and the tobacco EF-1A gene was used as an internal control. All experiments were repeated in triplicate. T1 GM plants (50-day-old) harvested from one T0 GM plant that had the highest dsPsFAR expression were used for rearing newly-emerged third-instar nymphs. The cultivation process used for T1 tobacco plants was the same as that used for tomato plants. Plants and mealybugs were reared under the same rearing conditions as described above.
Assessing RNAi knockdown of PsFAR in P. solenopsis
RT-qPCR was performed to assess the effects of RNAi. For the dsPsFAR and dsGFP microinjection groups, seven P. solenopsis individuals were collected 3 days after injection from each group and used for RT-qPCR. For the groups reared on WT and GM tobacco plants, 10 third-instar nymphs already feeding on the plants for 5 days were collected for RT-qPCR analysis. Primers for PsFAR I and PsFAR II were cited from Li et al.28. Siblings were used for developmental observation and survival quantification. All experiments were performed in triplicate.
Extraction and quantification of CHCs in P. solenopsis wax
CHCs in P. solenopsis wax was extracted from adult females that were collected 4 days after injection, or after feeding on GM tobacco for 9 days, following a procedure modified from Li et al.20,21. Briefly, 50 females (approximately 15 mg) were placed in a clean chromatography vial and immersed in 200 μl n-hexane. After 3 min of sonication in an ultrasonic vibrator (S06H, Zealway, China), the solvent was drawn into a new clean chromatography vial. This procedure was repeated twice, and finally, 200 μl of hexane was used to rinse the nymphs and vial. All hexane extracts were combined, followed by 20 min of sonication. Undissolved impurities were pelleted by 10 min of centrifugation at 10,000 rpm, the supernatant was dried absolutely under high-purity nitrogen gas, then re-suspended in 30 μl of hexane. After adding 300 ng n-heneicosane (C21) as an internal standard, samples were analyzed on the GC-MS system. The constant flow of helium was 1 ml/min. Splitless injection of 1.0 μl was made into a 30 m × 0.25 mm × 0.25 μm HP-5MS column (Agilent Technologies, Santa Clara, USA). The temperature program was operated as follows: 50 °C for 4 min, then 10 °C/min to 310 °C, hold 10 min. Injector and detector temperatures were respectively set at 270 °C and 300 °C. Mass detection was run under an EI mode with a 70 eV ionization potential and an effective m/z range of 45–650 at a scan rate of 5 scan/s. A C7–C40 n-alkanes standard (Sigma-Aldrich, MO, USA) was analyzed using the same conditions. Chemical compounds were identified by mapping against the NIST database and calibrated against a standard. The peak area was determined by the Agilent MassHunter system. The relative content of each n-alkane was quantified using the following formula: peak area of n-alkane/peak area of n-heneicosane (C21)*300. Six replicates for each treatment were performed.
Transmission electron microscopy (TEM) of P. solenopsis integument
To distinguish which pool of surface lipids was affected by PsFAR, we perform TEM to analyze the envelope integrity for adult females (24 h post-emergence, n = 5) sampled from both RNAi treated and control groups. The dissected integuments were first fixed in 2.5% glutaraldehyde overnight and rinsed three times with 0.1 M PBS (pH 7.0), 15 min for each time. After fixing with 1% osmium tetroxide for 1.5 h and rinsing twice with 0.1 M PBS (pH 7.0), samples were respectively dehydrated in an ethanol series (30 %, 50%, 70%, 80%, 90% and 95% (v/v)) for 15 min, followed by 20 min of dehydration in 100% ethanol and 100% acetone, respectively. Next, samples were impregnated (embedding agent: acetone 1:1, 1 h; embedding agent: acetone 3:1, 3 h; and pure embedding agent, 12 h), and embedded in Spurr resin (SPI-CHEM, USA) overnight at 70 °C. The ultrathin sections (70 nm) were prepared using an ultramicrotome (Leica, German). Following 0.1 M lead citrate staining, the samples were finally examined using TEM (JEM-2100plus, JEOL, Japan) operating at 200 kV of acceleration voltage.
Desiccation assay
To perform the desiccation tolerance bioassay, drying tubes with RH < 10% were prepared by putting approximately 10 g of packed fresh silica gel into a 50-ml centrifuge tube. In total, 30–34 adult females 4 days post injection or after feeding on GM tobacco for 9 days were put into a drying tube without food. In control experiments, females were starved at 75% RH in a normal 50-ml centrifuge tube. The humidity was assessed by a hygrometer (TH40W-EX, Miaoxin, Wenzhou, China). The rearing temperature was 27 ± 1 °C and the photoperiod was 14:10 (L:D). The number of surviving individuals was counted daily until all individuals died, and dead individuals were removed from the tube. Each treatment was performed in triplicate.
Waterproofing assay
For microinjected mealybugs, 30 adult females 4 days post-injection were collected and maintained in a rearing box. 500 μl of water was sprayed 10 cm straight down using a mini-sprayer (1.4 cm in diameter and 11.5 cm in height) (Supplementary Fig. 9) daily for 10 days. For mealybugs fed on GM tobacco plants, water was sprayed on plants directly using the same spray method. Both adaxial and abaxial surfaces of leaves harboring mealybugs were sprayed with water. The number of surviving P. solenopsis was recorded daily. The assay was repeated in triplicate.
Insecticide assay
To determine an appropriate concentration of insecticide for tolerance bioassays, groups of 30 one-day-old adult P. solenopsis females were collected from tomato or WT tobacco plants and placed in plastic boxes (same size as rearing box) that had been laid with a piece of filter paper at the bottom. For each box, 500 μl of an aqueous solution containing 25, 2.5 g/L, 250, 25, 2.5, and 0 mg/L of deltamethrin emulsion respectively were sprayed 10 cm straight down using the aforementioned mini-sprayer. One box with no treatment was set as the blank control. To reduce the effects of gastric toxicity, mealybugs were transferred into new rearing boxes immediately after spraying. The number of surviving mealybugs was counted three times within a 24 h interval. Each treatment was repeated in triplicate.
Based on the results obtained from the above assay, the 25 mg/L deltamethrin emulsion was used to evaluate the effects of PsFAR knockdown on insecticide resistance by P. solenopsis. The spray was performed on adult females 4 days post injection or after feeding on GM tobacco for 9 days following the same steps above. The surviving number of mealybugs was recorded 48 h after spraying. Assays for each treatment were performed in triplicate.
Statistics and reproducibility
All data sets were presented as mean ± SEM. Data were analyzed for statistical significance using analysis of variance (ANOVA) followed by Tukey’s multiple comparison test (Figs. 2c, 3d, Supplementary Figs. 3 and 8), and using student’s t-test (Figs. 4a, c, 5a, 6, Supplementary Figs. 5 and 7). Statistical significance is indicated with p-values as follows: *P < 0.05 and **P < 0.01. SPSS 19 and GraphPad Prism 8.0 were used for analyses.
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Source: Ecology - nature.com
