Insect culture

The insect culture of C. infuscatellus was obtained from the Sugarcane Farm, Hainan, China. The new field-collected insects were introduced every year in reared insect colonies to maintain genetic diversity at the Institute of Tropical Bioscience and Biotechnoloy, Hainan, China. The insects were reared on sugarcane plants at 25 ± 2 ^°C and 14:10 L:D photoperiod in insect rearing cages. The number of insects in each cage varied between 10 and 15. Newly planted sugarcane plants were provided twice each week. Egg masses were collected daily and stored in a petri dish with water-soaked filter paper and kept in an incubator (25 ± 2 ^°C, 70% RH, 16:8 L:D). The young larvae were fed with fresh sweet corn after they emerged in the same petri dishes.

Cloning of CiHR3 gene fragment and phylogenetic analysis

The CiHR3 gene from C. infuscatellus was the target gene for RNAi. The total RNA was prepared from the last instar stage of C. infuscatellus with Trizol reagent (Invitrogen Life Technologies,  www.invitrogen.com). The first strands of cDNAs were synthesized by a PrimeScript™ 1^st strand cDNA synthesis kit (Takara,  www.takarabio.com). A pair of degenerate primers CiHR3-middle fragment-P1 : 5′- ACA GWG GTG AAC TAC CAG TG -3′, and CiHR3-middle fragment-P2:5′- GAC CAT GRA ATT GGT CGC T -3′ were designed to amplify the homologous nucleic acid sequence by utilizing Primer Premier 5.0 (Premier Biosoft,  www.premierbiosoft.com). The primers were based on conserved amino acid regions found in lepidopteran insects. PCR was performed with the following conditions: 94 ^°C for five min, 35 cycles at 94 ^°C for 30 sec, 50 ^°C for 30 sec, and 72 ^°C for two min, and a final extension at 72 °C for 10 min. The amplified fragment was subcloned into the pMD19-T vector using the TA Cloning kit (Takara,  www.takarabio.com), and was subsequently transformed into Escherichia coli Top 10 competent cells (TransGen Biotechnologies,  www.transgen.com.cn). Nucleotide sequences were determined with an ABI PRISM 3730XL DNA Analyzer using a BigDyeTerminator v3.1 Cycle Sequencing Kit (Applied Biosystems,  www.biocompare.com). To clone a longer cDNA sequence, 3′ RACE was performed using gene specific primers for CiHR3 and adaptor primers supplied with Takara 3′ RACE cDNA Amplification Kit. For 3′ RACE, the gene specific primers that were designed based on the partial amplified fragment mentioned above were: forward specific primer, CiHR3-3′ RACE-GSP1: 5′ -CGG GTC AAC AGG AAC AGG TGT CA -3′, and nested specific primer, CiHR3-3′ RACE-GSP2: 5′- TGT CCA AGA AAC AAC GGG AGA AG -3′. Searching of similar sequences was performed using BlastP in the non-redundant protein sequences database of the NCBI website ( www.ncbi.nlm.nih.gov/). It showed homology with 15 related insect molt-regulating transcription factors from different insects. Multiple alignments of the deduced amino acid sequences were conducted by using Clustal X 1.81 software. A phylogenetic tree was constructed by using MEGA4.0.2 software ( www.megasoftware.net/) applying the method of Neighbor-Joining (NJ).

Construction of prokaryotic vector for expression of dsRNA

Before construction of the expression vector, the functional domain of the CiHR3 gene was predicted by using NCBI protein BLAST, especially ligand-binding domain (LBD), which is necessary to maintain insect growth and development (Montagne et al. 2010). For the CiHR3 target gene, two interference fragments were constructed viz., fragment one (F1 and R1), and fragment two (F2 and R2). Enhanced Green Fluorescent Protein (EGFP) gene served as a negative control (F3 and R3). The vector L4440 was used with host strain E. coli HT115. All PCR products of interference fragments and L4440 vector were digested with XhoI and KpnI and ligated using T4 DNA ligase at 16 ^°C overnight. The positive clones were then selected and sequenced.

Synthesis of dsRNA in bacteria

Total RNA was isolated from the last instar stage of C. infuscatellus using Trizol reagent (Invitrogen Life Technologies,  www.invitrogen.com). Then, two target fragments of 300–600 bp were amplified using gene-specific primers (Table 1) designed on the basis of CiHR3 gene sequences of C. infuscatellus. Similarly, primers of EGFP gene (negative control) were also designed (Table 1). The PCR products were first ligated to pMD19-T simple vector and then digested with XhoI and KpnI in order to construct recombinant vectors L4440-CiHR3-I1, L4440-CiHR3-I2, and L4440-EGFP. An empty L4440 vector consisted of two convergent T7 polymerase promoters in opposite orientations. The L4440 vector containing PCR products was transformed into competent HT115 (DE3) cells, an RNase III deficient E. coli strain with IPTG-inducible T7 polymerase activity. The single colonies of E. coli containing L4440 vector with insert were inoculated into LB medium containing ampicillin and cultured overnight. The bacterial solution was diluted to 100× with LB medium and grown to OD₆₀₀ = 0.6. IPTG was added to a final concentration of 0.8 mM, and the culture was incubated with shaking for eight hours at 37 ^°C. The solution was then sonicated on ice for 20 min and stored at -20 ^°C until use.

Table 1.

Annotation, GenBank database accession numbers, primers for synthesis of dsRNA, and sizes of interference fragments tested in this study.

Identification of dsRNA produced in E. coli HT115

To analyze the dsRNA synthesized in the bacteria, HT115 (DE3), total RNA was extracted from the bacterial cells using TRI reagent (Sangon Biotech,  www.sangon.com). The RNA was treated with DNase I (Takara,  www.takarabio.com) to remove the DNA contamination. The nucleic acid pellets were re-suspended in double distilled water and loaded onto a 1.5% agarose/TBE gel. The gel was stained with nucleic acid stain (Goldview®, SBS Genetech Co., Ltd.,  www.sbsbio.com) and photographed. Sample volumes were normalized according to the concentration of the RNA, and about 4 µg of total RNA was loaded into each lane of the gel.

Synthesis of dsRNA in vitro

The dsRNAs were also synthesized in vitro using the ScriptMAX™ Thermo T7 Transcription Kit (TOYOBO,  www.toyoboglobal.com). The recombinant plasmids L4440-CiHR3-I1, L4440-CiHR3-I2, and L4440-EGFP were extracted from the HT115 (DE3) cells with AxyPrep™ Plasmid Miniprep Kit (Axygen,  www.axygen.com). A high-fidelity DNA polymerase (Takara,  www.takarabio.com) was used to conduct PCR using the plasmid DNA and T7 (TAA TAC GAC TCA CTA TAG GG) primer. PCR conditions were 94 ^°C for 3 min followed by 35 cycles of 94 ^°C for 30 sec, 55 ^°C for 30 sec, and 72 °C for 1 min, and a final extension step at 72 ^°C for 10 min. PCR products were purified using a PCR purification kit (Tiangen Biotech,  www.tiangen.com) and used as a template for synthesis of dsRNA. The quality of the dsRNA was tested by electrophoresis and quantified using a spectrophotometer (NanoDrop Technologies,  www.nanodrop.com). The dsRNA was diluted in RNase-free H₂O to 4–5 µg/µL and stored at -80 ^°C before use.

Insect bioassay

The dsRNA-mediated gene silencing was conducted by voluntary feeding bioassays. Third and fifth instar C. infuscatellus larvae were starved for 12 hours before the initiation of feeding bioassays. Bioassays were carried out in two groups. One group was treated with bacterial-expressed dsRNA and the other group was treated with in vitro synthesized dsRNA. For each group, the bioassays were carried out in four treatments viz., L4440-CiHR3-I1, L4440-CiHR3-I2, L4440-EGFP, and water with three replications. The seven larvae with similar body size and weight were taken in one petri dish for each replication. Either 200 µL of bacterial expressing dsRNA or 50 µg of in vitro synthesized dsRNA (diluted in 200 µL double-distilled water for each delivery) was spread on fresh and tender corn kernels (with the similar weight in all treatments) in each petri dish. After treatment, petri dishes were placed in an incubator (25 – 2 ^°C, 70% RH, 16:8 L:D). All bioassays were carried out for seven days. The insects were treated for six consecutive days. On each day, insects from one petri dish were gently transferred with soft hair brush to a new petri dish containing treated fresh and tender corn kernels. The average body weight of larvae was recorded for 1–7 days in both groups. The EGFP dsRNA served as a negative control and water treatment served as a control for bacterial expressed dsRNA experiments. For in vitro dsRNA experiments, controls were fed with the EGFP dsRNA.

Quantitative real-timePCR (qRT-PCR) and reference gene selection

qRT-PCR was performed in an iCycler iQ RT-PCR detection system with iQ™ SYBR® Green supermix (Bio-Rad,  www.biorad.com). The Trizol reagent (Invitrogen Life Technologies,  www.invitrogen.com) was used to isolate total RNA from three individual insects on the 7^th day after treatment. The total RNAs were treated with DNase I (Takara,  www.takarabio.com). cDNA was synthesized using PrimeScript™ cDNA synthesis kit (Takara,  www.takarabio.com). DNase I-treated total RNA was used as a template. Each qRT-PCR reaction mixture (10 µL volume) consisted of 5 µL of FastStart SYBR Green Master (Roche Diagnostics,  www.roche.com), 1.2 µL of cDNA, and 0.6 µL each of forward and reverse gene-specific primers. An initial incubation was 95 ^°C for 3 min, followed by 40 cycles of 95 ^°C for 10 sec, 55 ^°C for 20 sec, and 72 ^°C for 30 sec, and a final extension at 72 ^°C for 10 min. A fluorescence reading determined the extension of amplification at the end of each cycle. Each experiment was repeated three times using samples from independent treatments. The similar quantitative primers were used for the two interference fragments, EGFP as a negative control, and water as a control: CiHR3-qRT-PCR-P1: 5′- AAC CAC CGC CAC AGC AGC CTT AC -3′; CiHR3-qRT-PCR-P2: 5′- GAT GTC TCC CTC CGC GTG ACC AA -3′. To detect mRNA level of CiHR3 gene, the β-actin gene of C. infuscatellus was evaluated with Bestkeeper software package. The primers for β-actin gene (GenBank no. JN985905) were: Ci-β-actin-P1: 5′- ACC AAC TGG GAC GAT ATG GAG AA -3′, Ci-β-actin-P2: 5′- CCT CAG TCA AGA GGA CTG GGT GC -3′. The relative expression levels for specific genes as compared to the β-actin reference gene were calculated by the 2_ΔΔCT _{method as} described by Livak and Schmittgen (2001).

Statistical analysis

Statistical analysis was carried out using SPSS software (SPSS v.13.0,  www.brothersoft.com). The knockdown efficiency test and the overall effects of RNAi on the growth and development of insects were analyzed using Student's t-test. The means were separated by using Duncan multiple mean separation techniques.

CiHR3 gene serves as an interference fragment

CiHR3 gene belongs to the family of molt-regulating transcription factors. The molting process in C. infuscatellus is regulated by the hormone-mediated cascade reaction consisting of a series of genes, and ecdysone regulation of transcription factor is a direct product of early induced gene expression (Zhao et al. 2004). The expression of this gene was immediately activated after a specific sequence on its target DNA was bound and directly involved in the late ecdysone-induced gene regulated by a molting cascade in the key factor. A phylogenetic tree constructed from the amino acid sequences of CiHR3 and other 15 related insect molt-regulating transcription factors indicated that CiHR3 is closely related to HR3 genes from lepidopteron insects, especially Galleria mellonella (Figure 1).

Verification of dsRNA synthesis in bacteria

The PCR products of two interference fragments and EGFP gene as a negative control were cloned into L4440 vector digested by XhoI and KpnI, separately. The resulting plasmids were transformed into HT115 (DE3) competent cells. After adding IPTG into the bacterial growth medium, the dsRNA of the target gene was induced. Thus, dsRNAs of all target gene fragments were successfully synthesized in the bacteria (Figure 2).

The effects of feeding dsRNAs on larval growth of C. infuscatellus

The effects of target genes on C. infuscatellus by feeding bacterial-expressed dsRNA were tested. The phenotypic effects were observed as a decrease in larval body weight. The body weight of the 3^rd instar larvae of C. infuscatellus was significantly decreased when feeding on bacteria-expressed CiHR3 dsRNA as compared with that of negative control or water treatment, and 25% insects died before entering into the 4^{th instar stage (Figure 3). Feeding CiHR3 dsRNA to the 5th} instar larvae of C. infuscatellus caused larval body weight to significantly decrease and 20% insects failed to enter into the pupal stage (Figure 4). However, there were no significant differences in the decrease in body weight between the treatments with bacterial expressed dsRNA and in vitro synthesized EGFP dsRNA (negative control) (Figure 5). Two interference fragments of CiHR3 gene were selected, viz., CiHR3-I1 and CiHR3-I2. The CiHR3-I2 fragment represents LBD domain of CiHR3 gene. The bioassay results indicated that feeding of CiHR3-I2 dsRNA was more effective than that of CiHR3-Il dsRNA, but there was no significant difference between them.

Knockdown efficiency after feeding bacteria-expressed and in vitro synthesized dsRNAs

To test the molecular effect of feeding RNAi on target-gene expression, qRT-PCR was conducted with individual samples at seven days after feeding with the bacteria-expressed dsRNA (Figure 6a) or in vitro synthesized dsRNA (Figure 6b). For bacterial expressed dsRNA experiments, expression of target genes was reported as the proportion of that in control samples fed with bacterial-expressed dsRNA. For in vitro dsRNA experiments, expression of target genes was calculated as the ratio of that in controls fed with the dsRNA using reporter EGFP gene as a template. All expression levels were normalized to β-actin. The common β-actin housekeeping gene for qRT-PCR analysis was selected. β-actin was chosen as the reference gene to calculate relative expression levels of target genes because it showed the most stable expression among samples. The feeding of bacterially expressed CiHR3 dsRNA showed a more efficient knockdown than in vitro synthesized dsRNA.